Purified therapeutic nanoparticles and preparation methods thereof

ABSTRACT

Purified therapeutic nanoparticles are provided herein. Such nanoparticles comprise an active pharmaceutical ingredient and human serum albumin, wherein the weight ratio of human serum albumin to the active ingredient in the therapeutic nanoparticles is from 0.01:1 to 1:1, and wherein the nanoparticles are substantially free of free human serum albumin that is not incorporated in the nanoparticles. The present disclosure also provides pharmaceutical compositions that comprise the purified therapeutic nanoparticles and are also substantially free of free human serum albumin. Methods for preparing and using the purified therapeutic nanoparticles and compositions thereof are also provided.

TECHNICAL FIELD

The present disclosure relates to the pharmaceutical field, and moreparticularly, to nanoparticles comprising human serum albumin and ahydrophobic drug, and the preparation method thereof, and even moreparticularly, to albumin nanoparticles comprising specific physicalproperties and the preparation method thereof.

BACKGROUND

As an anticancer drug, paclitaxel acts as a microtubule inhibitor inmitosis, and plays an important role in polymerization and stabilizationof intracellular microtubule. At the stage of mitosis, paclitaxeldisables the separation of microtubules, so that cells are blockedbetween G2 and M phase. As a result, the fast-dividing tumor cells arearrested at the phase of mitosis by paclitaxel, leading to the death ofthe cells due to hindered replication. Paclitaxel has important clinicalactivity to various cancers (for example, breast cancer, ovarian cancer,lung cancer, and bladder cancer, etc.).

Due to poor water-solubility, however, the application of paclitaxel inhuman body is limited. In order to make paclitaxel suitable forintravenous injection, Bristol-Myers Squibb (BMS) has developed TAXOL®,in which a surfactant polyoxyethylene castor oil (CREMOPHOR® EL) andanhydrous alcohol are added together as co-solvent to enhance thesolubility of paclitaxel. Taxol has significant activity to ovariancancer, breast cancer, lung cancer, esophagus cancer, and head and neckcancer. However, it has been demonstrated that Taxol may inducetherapy-related toxicity, and significant acute and accumulativetoxicity, such as myelosuppression, febrile neutropenia andhypersensitivity etc. These side effects are related to the surfactantpolyoxyethylene castor oil used (Anantbhushan et al., Asia Pac J ClinOncol. 2013; 9:176-181). Based on clinical research reports andpost-marketing safety data, an overall incidence of hypersensitivityinduced by Taxol is about 39%. At present, antihistamines and steroidsare administrated to patients ahead of time to alleviate the sideeffects due to the surfactant, when Taxol is used.

In order to improve the water solubility of paclitaxel, structuremodifications are also conducted by researchers using functional groupswhich may provide higher water solubility, for example, sulfonatederivatives (Kingston et al., U.S. Pat. No. 5,059,699 (1991)), andamino-acid ester derivatives (Mathew et al., J. Med. Chem. 35:145-151(1992)). They exhibit obvious biological activities after modification.However, these modifications may induce undesired side effects or reducethe pharmaceutical efficiency besides the increase of cost of thepharmaceutical formulations.

To avoid adverse effects of CREMOPHOR® EL in paclitaxel formulations,another drug delivery system that does not contain any emulsifying agentor surfactant was developed. Such a system is in the form ofmicro-particles or nanoparticles and contains albumin, a portion ofwhich forms complexes with paclitaxel. However, this system still hasmany disadvantages. For example, the formulation requires a large amountof human serum albumin (HSA), which may cause allergy. HSA is stillobtained from human blood, and has potential safety risks due topossible contaminations during blood collection and storage. Inaddition, HSA is relatively expensive and may be in shortage in certainregions.

SUMMARY

The present disclosure provides purified therapeutic nanoparticles,compositions comprising such nanoparticles, methods of preparing suchnanoparticles or compositions, and methods of using such nanoparticlesor compositions.

In one aspect, the present disclosure provides purified therapeuticnanoparticles that comprise an active ingredient and human serumalbumin, wherein the weight ratio of human serum albumin (HSA) to theactive ingredient in the therapeutic nanoparticles is selected from0.01:1, 0.02:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.10:1,0.11:1, 0.12:1, 0.13:1, 0.14:1, 0.15:1, 0.16:1, 0.17:1, 0.18:1, 0.19:1,0.2:1, 0.21:1, 0.22:1, 0.23:1, 0.24:1, 0.25:1, 0.26:1, 0.27:1, 0.28:1,0.29:1, 0.3:1, 0.31:1, 0.32:1, 0.33:1, 0.34:1, 0.35:1, 0.36:1, 0.37:1,0.38:1, 0.39:1, 0.4:1, 0.41:1, 0.42:1, 0.43:1, 0.44:1, 0.45:1, 0.46:1,0.47:1, 0.48:1, 0.49:1, 0.5:1, 0.51:1, 0.52:1, 0.53:1, 0.54:1, 0.55:1,0.56:1, 0.57:1, 0.58:1, 0.59:1, 0.6:1, 0.65:1, 0.70:1, 0.75:1, 0.8:1,0.85:1, 0.9:1, 0.95:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1,5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1 or the range between anytwo ratios above, and wherein the therapeutic nanoparticles aresubstantially free of free HSA that is not incorporated in thenanoparticles.

In certain embodiments, the weight ratio of human serum albumin to theactive ingredient is selected from 0.03:1, 0.04:1, 0.05:1, 0.06:1,0.07:1, 0.08:1, 0.09:1, 0.10:1, 0.11:1, 0.12:1, 0.13:1, 0.14:1, 0.15:1,0.16:1, 0.17:1, 0.18:1, 0.19:1, 0.2:1, 0.21:1, 0.22:1, 0.23:1, 0.24:1,0.25:1, 0.26:1, 0.27:1, 0.28:1, 0.29:1, 0.3:1, 0.31:1, 0.32:1, 0.33:1,0.34:1, 0.35:1, 0.36:1, 0.37:1, 0.38:1, 0.39:1, 0.4:1, 0.41:1, 0.42:1,0.43:1, 0.44:1, 0.45:1, 0.46:1, 0.47:1, 0.48:1, 0.49:1, 0.5:1, 0.6:1,0.7:1, 0.8:1, 0.9:1, or the range between any two ratios above.

In certain embodiments, the nanoparticles contain at most 5% free HSA(by weight).

In certain embodiments, the active ingredient has the properties ofbeing insoluble or slightly soluble in water, but soluble or freesoluble in an organic solvent.

In certain embodiments, the active ingredient is selected from taxanes,macrolides, camptothecins anthracycline antibiotics, colchicine,thiocolchicine dimer, amiodardone, liothyronine, cyclosporine,exemestane, flutamide, fulvestrant, romidepsin, semustine, andibuprofen.

In certain embodiments, the active ingredient is a taxane, such aspaclitaxel or docetaxel.

In certain embodiments, the organic solvent is a pure solvent having lowwater-solubility and low boiling point or its mixture with smallmolecular alcohols.

In certain embodiments, the active ingredient is encapsulated insidehuman serum albumin.

In certain embodiments, the average particle size of the therapeuticnanoparticles is selected from: 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 165, 170,175, 180, 185, 190, 195, 200 nm, or the range between any two numericalvalues above.

In another aspect, the present disclosure provides purified therapeuticnanoparticles that comprise an active ingredient and human serumalbumin, wherein the active ingredient is encapsulated inside humanserum albumin; the weight ratio of human serum albumin to the activeingredient is from 0.03:1 to 0.7:1; the average particle size of thetherapeutic nanoparticles is from 50 nm to 190 nm; and the nanoparticlesare substantially free of free HSA that is not incorporated in thenanoparticles.

In another aspect, the present disclosure provides a pharmaceuticalcomposition, comprising the purified therapeutic nanoparticles providedherein, wherein the composition is substantially free of free HSA thatis not incorporated in the nanoparticles.

In certain embodiments, the pharmaceutical composition is in the form ofliquid or lyophilized powder.

In certain embodiments where the pharmaceutical composition is in theform of lyophilized powder, it comprises one or more lyophilizationexcipients, such as mannitol, sucrose, lactose, maltose, trehalose,dextran, or a mixture thereof.

In certain embodiments, the pharmaceutical composition contains at most5% free HSA (by weight).

In another aspect, the present disclosure provides a method forpreparing the purified therapeutic nanoparticles provided herein. Themethod comprises:

1) dissolving the active ingredient in organic solvent to form an oilphase, and dissolving human serum albumin in water to form an aqueousphase;

2) forming an oil-in-water emulsion using the oil phase and aqueousphase above;

3) removing the organic solvent in the emulsion to obtain a suspensioncontaining the therapeutic nanoparticles; and

4) removing free HSA that is not incorporated in the nanoparticles fromthe suspension to obtain purified therapeutic nanoparticles.

In certain embodiments, the organic solvent is selected from one or moreof chloroform and ethanol.

In certain embodiments, the method further comprises: between steps 3)and 4), a step of dialyzing the suspension of step 3) with an aqueoussolution to remove remaining organic solvent from the suspension.

In certain embodiments, the aqueous solution is water.

In certain embodiments, said separating in step 4) is conducted using amethod selected from: centrifugation, dialysis, and exclusionchromatography.

In a related aspect, the present disclosure provides method forpreparing the pharmaceutical composition provided herein, comprising:

1) dissolving the active ingredient in organic solvent to form an oilphase, and dissolving human serum albumin in water to form an aqueousphase;

2) forming an oil-in-water emulsion using the oil phase and aqueousphase above;

3) removing the organic solvent in the emulsion to obtain a suspensioncontaining the therapeutic nanoparticles;

4) removing free HSA that is not incorporated in the nanoparticles toobtain purified therapeutic nanoparticles;

5) re-suspending the purified therapeutic nanoparticles in anexcipient-containing solution; and

6) optionally lyophilizing the re-suspension of the purified therapeuticnanoparticles to obtain the pharmaceutical composition.

In certain embodiments, the method further comprises: between steps 3)and 4), a step of dialyzing the suspension of step 3) with an aqueoussolution to remove remaining organic solvent from the suspension.

In certain embodiments, the aqueous solution is water.

In another related aspect, the present disclosure provides a method forpreparing the pharmaceutical composition provided herein, comprising:

1) dissolving the active ingredient in organic solvent to form an oilphase, and dissolving human serum albumin in water to form an aqueousphase;

2) forming an oil-in-water emulsion using the oil phase and aqueousphase above;

3) removing the organic solvent in the emulsion to obtain a suspensioncontaining the therapeutic nanoparticles;

4) dialyzing the suspension obtained after removal of the organicsolvent by an excipient-containing solution to remove free HSA that isnot incorporated in the nanoparticles; and

5) optionally lyophilizing the dialyzed suspension to obtain thepharmaceutical composition.

In certain embodiments, the method further comprises: between steps 3)and 4), a step of dialyzing the suspension of step 3) with an aqueoussolution to remove remaining organic solvent from the suspension.

In certain embodiments, the aqueous solution is water.

In another aspect, the present disclosure provides a method for treatingcancer, comprising: administrating a therapeutically effective amount ofthe pharmaceutical composition provided herein to a subject in needthereof.

In another aspect, the present disclosure provides a pharmaceuticalcomposition, comprising the therapeutic nanoparticles provided herein,wherein the composition further comprises HSA as a lyophilizationexcipient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Scanning electron microscopic image of the sample of Example 5.

FIG. 2. Scanning electron microscopic image of the nanoparticlesprepared using purified from the sample of Example 5.

FIG. 3. X diffraction pattern of paclitaxel.

FIG. 4. X diffraction pattern of lyophilized powder of albumin.

FIG. 5. X diffraction pattern of the paclitaxel-albumin nanoparticlescorresponding to Example 2.

FIG. 6. X diffraction pattern of the paclitaxel-albumin nanoparticlescorresponding to Example 4.

FIG. 7. X diffraction pattern of the paclitaxel-albumin nanoparticlescorresponding to Example 6.

FIG. 8. X diffraction pattern of the paclitaxel-albumin nanoparticlescorresponding to Example 8.

FIG. 9. In vitro release profiles of purified nanoparticles of variousformulations.

FIG. 10. In vitro release profiles of purified nanoparticles andtraditional formulations.

FIG. 11. Pharmacokinetic profiles of purified nanoparticles andtraditional formulations in dogs.

FIG. 12. Albumin concentration influences inhibition effect of drugs onMCF-7 cells.

FIG. 13. Albumin concentration influences inhibition effect of drugs onSPC-A-1 cells.

FIG. 14. Albumin concentration influences uptake of drugs by humanvascular endothelial cell EA.hy 926.

DETAILED DESCRIPTION

The present disclosure provides purified HSA-containing therapeuticnanoparticles, compositions comprising such nanoparticles and methods ofpreparing or using such nanoparticles and compositions.

The purified therapeutic nanoparticles or the compositions thatcomprising such nanoparticles have one or more of the following superiorproperties:

(1) Compared with previously known compositions that compriseHSA-containing nanoparticles, the purified therapeutic nanoparticles orcompositions thereof reduce allergic reactions in subjects to which thenanoparticles or compositions are administered (see e.g., Examples 25,27, and 61). While not wishing to be bound by any hypothesis, it isproposed that the reduction in allergic reactions may be resulted fromthe reduced amount of HSA polymers in the purified nanoparticles orcompositions thereof. It was discovered by the present inventors thatduring the preparation of nanoparticles, a portion of HSA monomersformed HSA polymers, causing more severe allergic reactions (see e.g.,Examples 32 and 60). Purification of nanoparticles from the initialpreparation eliminates most free HSA that is not incorporated intonanoparticles, including free HSA polymers.

(2) Compared with previously known compositions that compriseHSA-containing nanoparticles, certain compositions comprising purifiedtherapeutic nanoparticles provided herein are more stable (see e.g.,Examples 62 and 63). This is unexpected in view of the significantreduction in the ratio of HSA to active ingredients in the purifiednanoparticles and compositions of the present disclosure and in view ofthe belief in the art that a large amount of HSA is important orrequired for stabilizing compositions that comprise nanoparticles.

(3) While maintaining in vitro release profiles, maximum tolerateddoses, pharmacokinetic properties, and effectiveness in animal studies(see e.g., Examples 22 and 26-31), purified therapeutic nanoparticles orcompositions thereof provided herein are easier to be delivered to ortaken up by human target cells (e.g., human tumor cells and humanvascular endothelial cells) and achieved better desirable effects inthose cells (see e.g., Examples 58 and 59).

Unless otherwise defined, all scientific terms used herein have the samemeaning as commonly understood by one of ordinary skills in the art towhich this disclosure belongs.

Although the number ranges and approximate parameter values are given ina broad range of the present disclosure, all numbers in the specificexamples are described as precise as possible. However, certain errorexists in any numerical values essentially, which may be resulted fromthe standard deviation during the measurement for each of them.Additionally, it should be understood that all ranges disclosed hereinencompass any and all possible sub-ranges contained therein. Forexample, it should be understood that the range “from 1 to 10” describedherein encompasses any and all possible sub-ranges between the minimum 1and the maximum 10 (including the endpoints); i.e., all sub-rangesstarted from the minimum 1 or more, e.g., 1 to 6.1, and all sub-rangesended at the maximum 10 or less, e.g., 5.5 to 10. Additionally, itshould be understood that any reference referred as “incorporatedherein” is incorporated in its entirety.

Additionally, it should be noted that unless otherwise clearly andexplicitly stated, the singular form includes the plural referent, asused in the present disclosure. The term “or” and the term “and/or” areused interchangeably, unless otherwise clearly indicated in the context.

The term “nanoparticle” used herein refers to the particle with at leastone dimension (for example, 1, 2, or 3 dimensions) in nano-scale, forexample, at the level of about 1 nm, about 10 nm, or about 100 nm.

The term “about” means more or less than 10% of a particular value. Forexample, “about 50 nm” refers to 45 nm to 55 nm.

The term “therapeutic nanoparticle” used herein refers to thenanoparticles that can be used for the treatment or prevention ofdiseases, wherein the diseases, for example cancers, are preferablyselected from liver cancer, prostatic cancer and lung cancer.

The term “human serum albumin monomer” or “HSA monomer” used hereinrefers to the water soluble globulin composed of 585 amino acids and theapproximate molecular weight is around 66,000 Daltons. It is the mostabundant protein in human blood plasma. The retention time for humanserum albumin monomer is the longest in size exclusion chromatographyand count for the majority in normal human albumin products. HSA hasmultiple hydrophobic binding sites which can bind a diverse set ofdrugs, especially neutral and negatively charged hydrophobic compounds.

The term “human serum albumin polymer” or “HSA polymer” used hereinrefers to the sum of polymers polymerized by human serum albuminmonomer, including dimer, trimer and polymer. The retention time forhuman serum albumin polymer in size exclusion chromatography is shorterthan HSA monomers. HSA polymers are present in only a small amount(typically<5%) in human albumin products.

HSA is accumulated in various growing tumors and is used as a source ofenergy and amino acids uptake by tumor cells. gp60 (albondin) ishyper-expressed in the endothelium of blood vessels and binds HSA tocarry it into the underlying tissue by transcytosis. gp60 is notexpressed in tissue cells and it is not involved in the HSA transport intissue cells. SPARC (secreted protein, acidic and rich in cysteine), hasthe homologous amino acids sequences with gp60 and can bind HSA and gp60antibodies. SPARC is over-expressed in multiple tumor types and manystudies manifest that SPARC correlates with the tissue cellular uptakeof HSA. There are researches suggest that HSA-bound drugs also cross theendothelial barrier to the tumor tissue and internalize in tumor cellsvia Gp60-SPARC transmembrane transport pathway.

The term “substantially pure nanoparticles” or “purified nanoparticles”used herein refers to nanoparticles composed of human serum albumin andan active ingredient where less than 10% HSA (e.g., less than 9%, lessthan 8%, less than 7%, less than 6%, less than 5%, less than 4%, lessthan 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1%)is free HSA.

Similarly, the term “substantially free of free human serum albumin” or“substantially free of free HSA” used herein refers to having less than10% free HSA (e.g., less than 9%, less than 8%, less than 7%, less than6%, less than 5%, less than 4%, less than 3%, less than 2%, less than1%, less than 0.5%, or less than 0.1%) [78] The term “free human serumalbumin” or “free HSA” used herein refers to HSA is not incorporated innanoparticles. The amount of free HSA in nanoparticles or compositionsthereof may be measured by methods known in the art or a method providedin the present disclosure, such as in Examples 11 and 17-19.

The term “active ingredient” used herein refers to the activepharmaceutical ingredient. Particularly, the active ingredient refers toany substance or entity that can play a therapeutic role (for example,the treatment, prevention, alleviation or suppression of any diseaseand/or disorder).

The term “dialysis fold” used herein refers to the volume ratio ofdialysate consumed to a sample solution in a dialysis process where thevolume of the sample solution is kept constant.

The term “lyophilization excipient” used herein refers to compoundsadded to a pharmaceutical composition that comprises purifiedtherapeutic nanoparticles to maintain such nanoparticle during thefreeze-drying process.

The terms, “treat” and “treatment,” refer to medical management of adisease, disorder, or condition of a subject (i.e., patient) (see, e.g.,Stedman's Medical Dictionary). “Treating cancer” refers to reducing thenumber of symptoms of cancer, decreasing the severity of one or moresymptoms, or delaying cancer progression.

A “therapeutically effective dose” of a specific therapeutic agentrefers to that amount of the agent sufficient to result in reducing theseverity of, eliminating, or delaying the onset or reoccurrence of oneor more symptoms of cancer in a statistically significant manner.

In one aspect, the present disclosure provides purified therapeuticnanoparticles that comprise an active ingredient and HSA.

In some embodiments, the weight ratio of human serum albumin to theactive ingredient in the therapeutic nanoparticles is selected from thegroup consisting of 0.01:1, 0.02:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1,0.08:1, 0.09:1, 0.10:1, 0.11:1, 0.12:1, 0.13:1, 0.14:1, 0.15:1, 0.16:1,0.17:1, 0.18:1, 0.19:1, 0.2:1, 0.21:1, 0.22:1, 0.23:1, 0.24:1, 0.25:1,0.26:1, 0.27:1, 0.28:1, 0.29:1, 0.3:1, 0.31:1, 0.32:1, 0.33:1, 0.34:1,0.35:1, 0.36:1, 0.37:1, 0.38:1, 0.39:1, 0.4:1, 0.41:1, 0.42:1, 0.43:1,0.44:1, 0.45:1, 0.46:1, 0.47:1, 0.48:1, 0.49:1, 0.5:1, 0.51:1, 0.52:1,0.53:1, 0.54:1, 0.55:1, 0.56:1, 0.57:1, 0.58:1, 0.59:1, 0.6:1, 0.65:1,0.70:1, 0.75:1, 0.8:1, 0.85:1, 0.9:1, 0.95:1, 1:1, 1.5:1, 2:1, 2.5:1,3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1or the range between any two ratios above.

In some specific embodiments, the weight ratio of human serum albumin tothe active ingredient is selected from the group consisting of 0.03:1,0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.10:1, 0.11:1, 0.12:1,0.13:1, 0.14:1, 0.15:1, 0.16:1, 0.17:1, 0.18:1, 0.19:1, 0.2:1, 0.21:1,0.22:1, 0.23:1, 0.24:1, 0.25:1, 0.26:1, 0.27:1, 0.28:1, 0.29:1, 0.3:1,0.31:1, 0.32:1, 0.33:1, 0.34:1, 0.35:1, 0.36:1, 0.37:1, 0.38:1, 0.39:1,0.4:1, 0.41:1, 0.42:1, 0.43:1, 0.44:1, 0.45:1, 0.46:1, 0.47:1, 0.48:1,0.49:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1 or the range between any tworatios above, for example, 0.03:1 to 0.19:1, or 0.21:1 to 0.9:1.

More particularly, in certain embodiments, the weight ratio of humanserum albumin to the active ingredient is 0.043:1, 0.071:1, 0.13:1,0.15:1, 0.16:1, 0.17:1, 0.18:1, 0.24:1, or 0.57:1, or the range betweenany two ratios above, for example, 0.043:1 to 0.071:1, 0.043:1 to0.13:1, 0.043:1 to, 0.043:1 to 0.15:1, 0.043:1 to 0.16:1, 0.043:1 to0.17:1, 0.043:1 to 0.18:1, 0.043:1 to 0.24:1, 0.043:1 to 0.57:1, 0.071:1to 0.13:1, 0.071:1 to 0.15:1, 0.071:1 to 0.16:1, 0.071:1 to 0.17:1,0.071:1 to 0.18:1, 0.071:1 to 0.24:1, 0.071:1 to 0.57:1, 0.13:1 to0.15:1, 0.13:1 to 0.16:1, 0.13:1 to 0.17:1, 0.13:1 to 0.18:1, 0.13:1 to0.24:1, 0.13:1 to 0.57:1, 0.15:1 to 0.16:1, 0.15:1 to 0.17:1, 0.15:1 to0.18:1, 0.15:1 to 0.24:1, 0.15:1 to 0.57:1, 0.16:1 to 0.17:1, 0.16:1 to0.18:1, 0.16:1 to 0.24:1, 0.16:1 to 0.57:1, 0.17:1 to 0.18:1, 0.17:1 to0.24:1, 0.17:1 to 0.57:1, 0.18:1 to 0.24:1, 0.18:1 to 0.57:1 or 0.24:1to 0.57:1.

In some embodiments, the active ingredient suitable for encapsulationinside human serum albumin are insoluble or slightly soluble in water,and soluble or freely soluble in an organic solvent. The organic solventmay be a pure solvent with low water solubility (i.e., water solubilityless than 6%) and low boiling point (i.e., boiling point lower than 80°C.) or its mixture of the above-described pure solvent with smallmolecular alcohols including ethanol, tert-butanol, isopropanol, etc.The specific organic solvents include but are not limit to chloroform,dichloromethane, etc.

The active ingredient suitable for the present disclosure is taxanes,including but not limited to, paclitaxel, docetaxel, cabazitaxel,hydrophobic derivatives of docetaxel (e.g., 2′-O-hexanoyldocetaxel, and2′-benzoyldocetaxel); or macrolides, including but not limited torapamycin and its derivatives (e.g., temsirolimus and everolimus),epothilone B and its derivatives, tanespimycin and its derivatives; orcamptothecins, including but not limited to 10-hydroxy camptothecin,SN-38 and its derivatives; or anthracycline antibiotics, including butnot limited to aclacinomycin and pirarubicin; or other activeingredients including colchicine and its derivatives, thiocolchicinedimer, amiodardone, liothyronine, cyclosporine, exemestane, flutamide,fulvestrant, romidepsin, semustine, ibuprofen, cyclosporine, propofol,vinblastine, etc. In some embodiments, the active ingredient is selectedfrom one or more of paclitaxel and docetaxel. In particular embodiments,the active ingredient is paclitaxel. In some other embodiments, theactive ingredient is selected from docetaxel, rapamycin and itsderivatives, exemestane, flutamide, fulvestrant, etc.

In certain embodiments, the active ingredient includes but is notlimited to: chemotherapeutics, radiotherapeutic agents,immunotherapeutic agents, and thermally therapeutic agents etc. Forexample, aminoglutethimide, azathioprine, bleomycin sulphate, busulfan,carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporine,dacarbazine, dactinomycin, daunorubicin, amycin, paclitaxel, etoposide,fluorouracil, interferon-α, lomustine, mercaptopurine, methoptrexate,mitotane, procarbazine hydrochloride, thioguanine, vinblastine sulfateand vincristine sulfate.

In some embodiments, the average particle size of the therapeuticnanoparticles is selected from 30, 50, 70, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 165, 170, 175, 180, 185, 190, 195, 200 nm,or the range between any two numerical values above. It should beunderstood by the skilled person in the art that the particle size canbe determined using any appropriate method that exists or will appear inthe future, which includes but is not limited to settling method, sievemethod, microscopic observation or laser particle sizer. It also shouldbe understood that when the therapeutic nanoparticles disclosed in thepresent disclosure comprise multiple particles, not all therapeuticnanoparticles should have the same particle size, and they will also beencompassed in the scope of the present disclosure, as long as theiraverage particle size (i.e., the average particle diameter) falls in theabove range. In some specific embodiments, the particle size isdetermined by a laser particle sizer; and the average particle size ofthe therapeutic nanoparticles is selected from 50, 60, 70, 80, 90, 100,110, 120, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160 nm, or the range between anytwo numerical values above.

In some aspects, the particle sizes of the nanoparticles are in aspecific range, for example, from 30 nm to 200 nm, preferably, from 50to 190 nm. Preferably, the particle size is substantially uniform.

In one specific embodiment, purified therapeutic nanoparticles areprovided, which comprise paclitaxel and human serum albumin, whereinpaclitaxel is encapsulated in human serum albumin; and the weight ratioof human serum albumin to paclitaxel is 0.043:1, and the averageparticle size of the therapeutic nanoparticles is 140 nm.

In another specific embodiment, purified therapeutic nanoparticles areprovided, which comprise paclitaxel and human serum albumin, whereinpaclitaxel is encapsulated in human serum albumin; and the weight ratioof human serum albumin to paclitaxel is 0.071:1, and the averageparticle size of the therapeutic nanoparticles is 134 nm.

In another specific embodiment, purified therapeutic nanoparticles areprovided, which comprise paclitaxel and human serum albumin, whereinpaclitaxel is encapsulated in human serum albumin; and the weight ratioof human serum albumin to paclitaxel is 0.13:1, and the average particlesize of the therapeutic nanoparticles is 125 nm.

In another specific embodiment, purified therapeutic nanoparticles areprovided, which comprise paclitaxel and human serum albumin, whereinpaclitaxel is encapsulated in human serum albumin; and the weight ratioof human serum albumin to paclitaxel is 0.15:1, and the average particlesize of the therapeutic nanoparticles is 136 nm.

In another specific embodiment, purified therapeutic nanoparticles areprovided, which comprise paclitaxel and human serum albumin, whereinpaclitaxel is encapsulated in human serum albumin; and the weight ratioof human serum albumin to paclitaxel is 0.16:1, and the average particlesize of the therapeutic nanoparticles is 133 nm.

In another specific embodiment, purified therapeutic nanoparticles areprovided, which comprise paclitaxel and human serum albumin, whereinpaclitaxel is encapsulated in human serum albumin; and the weight ratioof human serum albumin to paclitaxel is 0.17:1, and the average particlesize of the therapeutic nanoparticles is 136 nm.

In another specific embodiment, purified therapeutic nanoparticles areprovided, which comprise paclitaxel and human serum albumin, whereinpaclitaxel is encapsulated in human serum albumin; and the weight ratioof human serum albumin to paclitaxel is 0.18:1, and the average particlesize of the therapeutic nanoparticles is 138 nm.

In another specific embodiment, purified therapeutic nanoparticles areprovided, which comprise paclitaxel and human serum albumin, whereinpaclitaxel is encapsulated in human serum albumin; and the weight ratioof human serum albumin to paclitaxel is 0.24:1, and the average particlesize of the therapeutic nanoparticles is 141 nm.

In another specific embodiment, purified therapeutic nanoparticles areprovided, which comprise paclitaxel and human serum albumin, whereinpaclitaxel is encapsulated in human serum albumin; and the weight ratioof human serum albumin to paclitaxel is 0.57:1, and the average particlesize of the therapeutic nanoparticles is 145 nm.

In another specific embodiment, purified therapeutic nanoparticles areprovided, which comprise docetaxel and human serum albumin, whereindocetaxel is encapsulated in human serum albumin; and the weight ratioof human serum albumin to docetaxel is 0.1:1, and the particle size ofthe therapeutic nanoparticles is in the range from 110 nm to 150 nm.

Purified therapeutic nanoparticles of the present disclosure aresubstantially free of free HSA. In certain embodiments, purifiedtherapeutic nanoparticles contain at most 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, 0.5% or 0.1% free HSA by weight (i.e., at most 8%, 7%, 6%, 5%, 4%,3%, 2%, 1%, 0.5% or 0.1% of total HSA in the purified therapeuticnanoparticles is free HSA that does not bind an active ingredient and isnot incorporated into nanoparticles.

In certain embodiments, purified therapeutic nanoparticles consistessentially of an active ingredient and HSA where substantially all ofthe HSA (i.e., more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5%, or 99.9% of HSA) are bound to the active ingredient.

In certain embodiments, purified therapeutic nanoparticles comprise aminimum amount (e.g. less than 0.05, 0.04, 0.03, 0.02, or 0.01 mg/ml, orless than 5, 1, 0.5, 0.1, 0.05, or 0.01 μg/ml) of one or more organicsolvents used in preparing such nanoparticles.

In certain embodiments, purified therapeutic nanoparticles do notcomprise any surfactants.

In certain embodiments, purified nanoparticles provided herein have arelatively high zeta potential, such as from −20 to −45 or from −25 to−40.

In another aspect, the present disclosure provides a pharmaceuticalcomposition that comprises purified therapeutic nanoparticles providedherein and is substantially free of free HSA that is not incorporated inthe nanoparticles. In certain embodiments, the pharmaceuticalcomposition comprises less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.5%, or 0.1% free HSA.

In some embodiments, the pharmaceutical composition is provided in theform of liquid, including but not limited to, the form suitable forinjection to a subject. In one specific embodiment, the pharmaceuticalcomposition is prepared into an injection.

In some other embodiments, the pharmaceutical composition is provided inthe form of solid, including, but not limited to, dry powder orlyophilized powder.

In certain specific embodiments, the nanoparticle or the pharmaceuticalcomposition of the present disclosure is free of deferoxamine or thesalt thereof, for example, deferoxamine mesylate.

In certain specific embodiments, the purified nanoparticles or thepharmaceutical compositions of the present disclosure are free ofadditional stabilizer.

When provided in a liquid form, the pharmaceutical composition comprisesthe therapeutic nanoparticles of the present disclosure and apharmaceutically acceptable carrier. The therapeutic nanoparticles aresuspended in the pharmaceutically acceptable carrier. Thepharmaceutically acceptable carrier includes but is not limited to abuffer solution, a preservative agent, water for injection, normalsaline, and an isotonic solution. In some specific embodiments, theconcentration of the active ingredient of therapeutic nanoparticles is1-10 mg/ml (e.g., 5 mg/ml) in the pharmaceutical composition in a liquidform.

When provided in a solid form, the pharmaceutical composition comprises,consists essentially of, or consists of: the therapeutic nanoparticlesof the present disclosure and a lyophilization excipient. In somespecific embodiments, the lyophilization excipient is selected from oneor more of mannitol, sucrose, lactose, maltose, trehalose, and dextran.In certain specific embodiments, the therapeutic nanoparticles aresuspended in a solution of lyophilization excipient with theconcentration from 5% to 10%, and subsequently the solution islyophilized to obtain a pharmaceutical composition in a form oflyophilized powder. In particular embodiments, the solution oflyophilization excipient is selected from one or more of 5% mannitolsolution, 10% sucrose solution, 5% dextran solution, 10% lactosesolution, 10% trehalose solution, and 10% maltose solution. In somespecific embodiments, the content of the therapeutic nanoparticles inthe pharmaceutical composition in a solid form is from 4.8% to 10% byweight.

The method for preparing purified therapeutic nanoparticles of thepresent disclosure is also provided, comprising: (1) preparing asuspension containing therapeutic nanoparticles by mixing an activeingredient with human serum albumin, and (2) purifying nanoparticlesfrom the suspension to obtain substantially pure therapeuticnanoparticles.

In another aspect, the present disclosure also provides therapeuticnanoparticles obtained by the method provided herein.

In another aspect, a method for preparing purified therapeuticnanoparticles is provided. The method comprises:

1) dissolving the active ingredient in organic solvent to form an oilphase, and dissolving human serum albumin in water to form an aqueousphase;

2) forming an oil-in-water emulsion using the oil phase and aqueousphase;

3) removing the organic solvent in the emulsion to obtain a suspensioncontaining the therapeutic nanoparticles;

4) removing free HSA that is not incorporated in the nanoparticles fromthe suspension to obtain purified therapeutic nanoparticles.

Appropriate organic solvent(s) can be selected by the skilled personbased on the properties of the active ingredient. In some specificembodiments, the suitable organic solvent is chloroform, ethanol or amixture of chloroform and ethanol when the active ingredient is taxanes.More particularly, the suitable organic solvent is a mixture ofchloroform and ethanol when the active ingredient is paclitaxel ordocetaxel. In some specific embodiments, the volume ratio betweenchloroform and ethanol is in the range from 1:1 to 20:1, and forexample, it is selected from 1:1, 4:1, 9:1 and 11:1. In some specificembodiments, the concentration of human serum albumin in the aqueousphase is in the range from 2% to 10% (w/v); for example, it is selectedfrom 2%, 4%, 5% and 10%. In some embodiments, the ratio between theactive ingredient and the organic solvent in the oil phase is in therange from 0.3-7.5 g/15-20 ml. In some specific embodiments, the ratiobetween the active ingredient and the organic solvent in the oil phaseis selected from: 0.3 g/15 ml, 0.6 g/15 ml, 1 g/20 ml, 1.25 g/15 ml, 1.8g/15 ml, 2 g/15 ml, 2.5 g/15 ml, 3 g/20 ml, 3 g/15 ml, 5 g/15 ml, 7.5g/15 ml, or the range between any two numerical values above.

When an oil-in-water emulsion is formed with the oil phase and theaqueous phase, the volume ratio between the two phases is selected from1:10 to 1:100. In some embodiments, the mixing ratio of the oil phaseand the aqueous phase is 3:100 or 1:25. An oil-in-water emulsion isformed using the method known in the art, which includes, but notlimited to, homogenization. In particular embodiments, the mixture ofthe oil phase and the aqueous phase is emulsified using a high sheardisperser, and subsequently, it is homogenized using a high pressurehomogenizer, so as to obtain an oil-in-water emulsion. In particularembodiments, the mixture of the oil phase and the aqueous phase isemulsified for 2-10 min using a high shear disperser, and subsequently,it is homogenized using a high pressure homogenizer under the pressureof 10000-20000 psi, so as to obtain an oil-in-water emulsion.

The organic solvent can be removed from the emulsion using anyappropriate method. In some embodiments, the organic solvent is removedfrom the emulsion using rotary vacuum evaporation. In particularembodiments, the organic solvent is removed from the emulsion using arotary evaporator at 40° C. under the pressure of 40 mbar. After removalof the organic solvent, the resulting suspension comprises thetherapeutic nanoparticles of the present disclosure. However, excessivealbumin, which does not participate in the formation of thenanoparticle, is also contained in the suspension.

Excessive free albumin is further removed from the suspension to obtainpurified therapeutic nanoparticles of the present disclosure that aresubstantially free of free HSA. In some embodiments, this is conductedusing centrifugation, dialysis, or exclusion chromatography. Inparticular embodiments, the suspension can be directly used for removingfree HSA after removal of the organic solvent. Alternatively, it can bestored for further use.

Since intravenous infusion is the desirable administration route for thetherapeutic nanoparticles of the present disclosure, the product must besterile. Heat sterilizing is not applicable in the present disclosure,since both the nanoparticles and human serum albumin are sensitive totemperature. Thus feasible sterilization methods include asepticproduction or sterilization filtration. In such cases, after removal ofthe organic solvent, the suspension is sterilized by passing through afilter, and subsequently, it is lyophilized to obtain a solid. The solidobtained is re-suspended in sodium chloride solution. In someembodiments, the solid is re-suspended in 0.9% sodium chloride solutionto reach a paclitaxel concentration of about 5 mg/ml.

When the therapeutic nanoparticles are purified using centrifugation,removal of free HSA is performed at 21000×g for 60 min or at equivalentconditions.

When the therapeutic nanoparticles are purified by dialysis, thenanoparticle-containing suspension obtained in step 3) is dialyzed usingan ultrafiltration membrane to remove free HSA. In particularembodiments, the nanoparticle liquid obtained according to the method ofthe present disclosure is dialyzed in equal volume of 5% mannitolsolution using a regenerated cellulose ultrafiltration membrane with thecutoff molecular weight of 300K, and the dialysis fold is 5.

When therapeutic nanoparticles are purified using exclusionchromatography, therapeutic nanoparticles are separated from free HSAusing an exclusion chromatographic column. In particular embodiments,nanoparticle-containing suspension obtained in step 3) is applied onto asepharose column, and the eluting peak corresponding to therapeuticnanoparticles is collected.

In certain embodiments, the method further comprises between steps 3)and 4) a step of dialyzing the suspension of step 3) with an aqueoussolution (e.g., water or 5% mannitol solution, 10% sucrose solution, 5%dextran solution, 10% lactose solution, 10% trehalose solution, and 10%maltose solution, etc) to remove the remaining organic solvent from thesuspension. For example, the suspension may be dialyzed in water or anaqueous solution using an ultrafiltration membrane that allows theorganic solvent to pass but not HSA or nanoparticles (e.g., a celluloseultrafiltration membrane with a cutoff molecular weight of 30K). Whilenot wishing to be bound by any theory, the removal or reduction of theremaining organic solvent prior to removal of free HSA from thenanoparticle-containing suspension may improve stability of purifiednanoparticles or compositions thereof.

In another aspect, a method for preparing a pharmaceutical compositioncomprising the therapeutic nanoparticles is also provided. The methodcomprises:

1) dissolving the active ingredient in organic solvent to form an oilphase, and dissolving human serum albumin in water to form an aqueousphase;

2) forming an oil-in-water emulsion using the oil phase and aqueousphase;

3) removing the organic solvent in the emulsion to obtain a suspensioncontaining the therapeutic nanoparticles;

4) removing free HSA that is not incorporated in the nanoparticles toobtain purified therapeutic nanoparticles;

5) re-suspending the purified therapeutic nanoparticles in apharmaceutically acceptable carrier-containing solution to obtain thepharmaceutical composition; and

6) optionally lyophilizing the re-suspension of the purified therapeuticnanoparticles where the pharmaceutical composition is in the form ofsolid.

Steps 1) and 4) of this method are the same as described with respect tothe method of preparing purified therapeutic nanoparticles. In addition,in certain embodiments, the method comprises between steps 3) and 4) astep of dialyzing the suspension of step 3) with an aqueous solution toremove the remaining organic solvent from the suspension also asdescribed with respect to the method of preparing purified therapeuticnanoparticles.

In some embodiments, the pharmaceutically acceptable carrier-containingsolution is the solution containing lyophilization excipient. In somespecific embodiments, the lyophilization excipient is selected from oneor more of mannitol, sucrose, lactose, maltose, trehalose, and dextran.In some other specific embodiments, the lyophilization excipient is HSA.In specific embodiments, the therapeutic nanoparticles are suspended ina solution of lyophilization excipient at the concentration from 5% to10%.

Optionally, the pharmaceutical composition is prepared into lyophilizedpowder after lyophilization. In particular embodiments, the solution oflyophilization excipient is selected from one or more of 5% mannitolsolution, 10% sucrose solution, 5% dextran solution, 10% lactosesolution, 10% trehalose solution, 10% maltose solution, and 10% humanserum albumin solution.

In some specific embodiments, the content of the therapeuticnanoparticles in the pharmaceutical composition in liquid form is from0.1% to 30%, and preferably, from 0.2% to 10%, and more preferably, from0.5% to 5%, for example, 1%. In some specific embodiments, the contentof the active ingredient (for example, paclitaxel) in the pharmaceuticalcomposition in liquid form of the present disclosure is from 0.1 to 100mg/ml, and preferably, from 0.5 to 50 mg/ml, and more preferably, from 1to 20 mg/ml, for example, 5 mg/ml.

In certain embodiments, the content of the therapeutic nanoparticles inthe pharmaceutical composition in solid form is from 0.1% to 80% byweight, and preferably, from 0.5% to 50%, and more preferably, from 1%to 30%, for example, from 2% to 10%.

In some specific embodiments, the content of the active pharmaceuticalingredient (for example, paclitaxel) in the pharmaceutical compositionin liquid form is from 0.1% to 80% by weight, and preferably, from 0.5%to 50%, and more preferably, from 1% to 30%, for example, from 2% to10%.

Alternatively, the pharmaceutical composition of the present disclosurecan be prepared using another procedure of dialysis. The methodcomprises:

1) dissolving the active ingredient in organic solvent to form an oilphase, and dissolving human serum albumin in water to form a aqueousphase;

2) forming an oil-in-water emulsion using the oil phase and aqueousphase above;

3) removing the organic solvent in the emulsion to obtain a suspensioncontaining the therapeutic nanoparticles;

4) dialyzing the suspension obtained after removal of the organicsolvent by a pharmaceutically acceptable carrier-containing solution toremove free HSA that is not incorporated in the nanoparticles; and

5) optionally lyophilizing the dialyzed suspension when thepharmaceutical composition is prepared in the form of solid.

Steps 1) and 4) of this method are the same as described with respect tothe method of preparing purified therapeutic nanoparticles when dialysisis used to remove free HSA. In addition, in certain embodiments, themethod comprises between steps 3) and 4) a step of dialyzing thesuspension of step 3) with an aqueous solution to remove the remainingorganic solvent from the suspension also as described with respect tothe method of preparing purified therapeutic nanoparticles.

In some embodiments, a pharmaceutically acceptable carrier-containingsolution is used as the dialysate, which is the lyophilizationexcipient-containing solution. In some specific embodiments, thelyophilization excipient is selected from one or more of mannitol,sucrose, lactose, maltose, trehalose, and dextran.

In specific embodiments, the therapeutic nanoparticles are suspended ina solution of lyophilization excipient with the concentration from 5% to10%. Optionally, the pharmaceutical composition is prepared intolyophilized powder after lyophilization. In particular embodiments, thesolution of lyophilization excipient is selected from one or more of 5%mannitol solution, 10% sucrose solution, 5% dextran solution, 10%lactose solution, 10% trehalose solution, 10% maltose solution, and 10%human serum albumin solution. In some embodiments, dialysis is performedusing an ultrafiltration membrane with cutoff molecular weight of 300 k.

In another aspect, the present disclosure provides methods for using thepurified therapeutic nanoparticles or compositions thereof. Because thepresent purified nanoparticles or compositions are effective means fordelivering various active ingredients, they may be used for treating anydiseases or disorders that are responsive to the active ingredients. Forexample, the purified therapeutic nanoparticles or compositions thereofmay be used in treating cancer, such as liver cancer, prostatic cancerand lung cancer. Additional diseases or disorders that may be treatedinclude breast cancer, multiple myeloma, transplant rejection, coloncancer, lymphoma, fever, etc.

In a particular aspect, the present disclosure provides a method fortreating cancer that comprises administering a therapeutically effectiveamount of a pharmaceutical composition provided herein to a subject inneed thereof. In specific embodiments, the subject is a mammal,including but not limited to human, canine, mouse, and rat.

A therapeutically effective amount of a pharmaceutical composition maybe determined or adjusted depending on various factors including thespecific therapeutic agents or pharmaceutical compositions, the routesof administration, the subject's condition, that is, stage of thedisease, severity of symptoms caused by the disease, general healthstatus, as well as age, gender, and weight, and other factors apparentto a person skilled in the medical art. Similarly, the dose of thetherapeutic for treating a disease or disorder may be determinedaccording to parameters understood by a person skilled in the medicalart. Optimal doses may generally be determined using experimental modelsand/or clinical trials.

The pharmaceutical composition may be administered via through anysuitable routes, for example, oral, nasal, intracutaneous, subcutaneous,intramuscular or intravenous administration.

In another aspect, a pharmaceutical kit is also provided in the presentdisclosure, which comprises purified therapeutic nanoparticles or apharmaceutical composition thereof provided herein. If required, thepharmaceutical kit also comprises instruction, package, and a containerholding the therapeutic nanoparticles or the pharmaceutical composition.

EXAMPLES

The Examples below were intended to better illustrate the therapeuticnanoparticles and the pharmaceutical composition disclosed herein, andnot to limit any aspect of the present disclosure.

Example 1

3 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech Co., Ltd) wasdissolved into 20 ml chloroform/ethanol (9:1, v/v), and added into 500ml human serum albumin solution (5% w/v) (CAS: 70024-90-7, GuangdongShuanglin Biopharmaceutical. Co., Ltd.). The mixture was emulsified for2 min using a high shear disperser (Model F22E, Fluko Co., Ltd.,Shanghai) to obtain a primary emulsion. The primary emulsion was thenhomogenized using a high pressure homogenizer (Model M110-EH30K, MFICCompany, USA) under pressure of 10000-20000 psi to obtain anano-emulsion. Subsequently, the nano-emulsion was transferred to arotatory evaporator (Model R-210, Buchi Company, Switzerland) to removethe organic solvent in the solution by vacuum evaporation at 40 mbar andat 40° C. in a water-bath. The paclitaxel-albumin nanoparticles werethus generated with an average diameter of 136 nm, and the suspensionwas translucent.

The suspension can be smoothly filtered through a 0.22 μm sterile filter(Sartorius AG, Germany). There was no significant variation of theparticle size after filtration, and no significant change was observedafter storage for 48 h at room temperature. The suspension was aliquotedand lyophilized for 24 h in a lyophilizer (Model LD-855, Millrock, USA)to obtain a stable off-white cake.

Example 2

0.32 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech Co., Ltd) wasdissolved into 15 ml chloroform/ethanol (11:1, v/v), and added intowhich 500 ml human serum albumin solution (4% w/v) (CAS: 70024-90-7,Guangdong Shuanglin Biopharmaceutical. Co., Ltd.). The mixture wasemulsified for 2 min using a high shear disperser (Model F22E, FlukoCo., Ltd., Shanghai) to obtain a primary emulsion. The primary emulsionwas then homogenized using a high pressure homogenizer (ModelM110-EH30K, MFIC Company, USA) under a pressure of 10000-20000 psi toobtain a nano-emulsion. Subsequently, the nano-emulsion was transferredto a rotatory evaporator (Model R-210, Buchi Company, Switzerland) toremove the organic solvent in the solution by vacuum evaporation at 40mbar and at 40° C. in a water-bath. The paclitaxel-albumin nanoparticleswere thus generated with an average diameter of 145 nm, and thesuspension was translucent.

The suspension can be smoothly filtered through a 0.22 μm sterile filter(Sartorius AG, Germany). There was no significant variation of theparticle size after filtration, and no significant change was observedafter storage for 48 h at room temperature. The suspension was aliquotedand lyophilized for 24 h in a lyophilizer (Model LD-85S, Millrock, USA)to obtain a stable off-white cake.

Example 3

0.63 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech Co., Ltd) wasdissolved into 15 ml chloroform/ethanol (11:1, v/v), and added into 500ml human serum albumin solution (4% w/v) (CAS: 70024-90-7, GuangdongShuanglin Biopharmaceutical. Co., Ltd.). The mixture was emulsified for2 min using a high shear disperser (Fluko FZ-20) to obtain a primaryemulsion. The primary emulsion was then homogenized using a highpressure homogenizer (Model M110-EH30K, MFIC Company, USA) under apressure of 10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator (Model R-210,Buchi Company, Switzerland) to remove the organic solvent in thesolution by vacuum evaporation at 40 mbar and at 40° C. in a water-bath.The paclitaxel-albumin nanoparticles were thus generated with an averagediameter of 141 nm, and the suspension was translucent.

The suspension can be smoothly filtered through a 0.22 μm sterile filter(Sartorius AG, Germany). There was no significant variation of theparticle size after filtration, and no significant change was observedafter storage for 48 h at room temperature. The suspension was aliquotedand lyophilized for 24 h in a lyophilizer (Model LD-85S, Millrock, USA)to obtain a stable off-white cake.

Example 4

1.25 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech Co., Ltd) wasdissolved into 15 ml chloroform/ethanol (11:1, v/v), and added intowhich 500 ml human serum albumin solution (4% w/v) (CAS: 70024-90-7,Guangdong Shuanglin Biopharmaceutical. Co., Ltd.). The mixture wasemulsified for 2 min using a high shear disperser (Model F22Z, FlukoCo., Ltd., Shanghai) to obtain a primary emulsion. The primary emulsionwas then homogenized using a high pressure homogenizer (ModelM110-EH30K, MFIC Company, USA) under a pressure of 10000-20000 psi toobtain a nano-emulsion. Subsequently, the nano-emulsion was transferredto a rotatory evaporator (Model R-210, Buchi Company, Switzerland) toremove the organic solvent in the solution by vacuum evaporation at 40mbar and at 40° C. in a water-bath. The paclitaxel-albumin nanoparticleswere thus generated with an average diameter of 138 nm, and thesuspension was translucent.

The suspension can be smoothly filtered through a 0.22 μm sterile filter(Sartorius AG, Germany). There was no significant variation of theparticle size after filtration, and no significant change was observedafter storage for 48 h at room temperature. The suspension was aliquotedand lyophilized for 24 h in a lyophilizer (Model LD-85S, Millrock, USA)to obtain a stable off-white cake.

Example 5

1.88 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech Co., Ltd) wasdissolved into 15 ml chloroform/ethanol (11:1, v/v), and added intowhich 500 ml human serum albumin solution (4% w/v) (CAS: 70024-90-7,Guangdong Shuanglin Biopharmaceutical. Co., Ltd.). The mixture wasemulsified for 2 min using a high shear disperser (Fluko FZ-20) toobtain a primary emulsion. The primary emulsion was then homogenizedusing a high pressure homogenizer (Model M110-EH30K, MFIC Company, USA)under a pressure of 10000-20000 psi to obtain a nano-emulsion.Subsequently, the nano-emulsion was transferred to a rotatory evaporator(Model R-210, Buchi Company, Switzerland) to remove the organic solventin the solution by vacuum evaporation at 40 mbar and at 40° C. in awater-bath. The paclitaxel-albumin nanoparticles were thus generatedwith an average diameter of 133 nm, and the suspension was translucent.

The suspension can be smoothly filtered through a 0.22 μm sterile filter(Sartorius AG, Germany). There was no significant variation of theparticle size after filtration, and no significant change was observedafter storage for 48 h at room temperature. The suspension was aliquotedand lyophilized for 24 h in a lyophilizer (Model LD-85S, Millrock, USA)to obtain a stable off-white cake.

Example 6

2.5 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech Co., Ltd) wasdissolved into 15 ml chloroform/ethanol (11:1, v/v), and added intowhich 500 ml human serum albumin solution (4% w/v) (CAS: 70024-90-7,Guangdong Shuanglin Biopharmaceutical. Co., Ltd.). The mixture wasemulsified for 2 min using a high shear disperser (Fluko FZ-20) toobtain a primary emulsion. The primary emulsion was then homogenizedusing a high pressure homogenizer (Model M110-EH30K, MFIC Company, USA)under a pressure of 10000-20000 psi to obtain a nano-emulsion.Subsequently, the nano-emulsion was transferred to a rotatory evaporator(Model R-210, Buchi Company, Switzerland) to remove the organic solventin the solution by vacuum evaporation at 40 mbar and at 40° C. in awater-bath. The paclitaxel-albumin nanoparticles were thus generatedwith an average diameter of 125 nm, and the suspension was translucent.

The suspension can be smoothly filtered through a 0.22 μm sterile filter(Sartorius AG, Germany). There was no significant variation of theparticle size after filtration, and no significant change was observedafter storage for 48 h at room temperature. The suspension was aliquotedand lyophilized for 24 h in a lyophilizer (Model LD-85S, Millrock, USA)to obtain a stable off-white cake.

Example 7

5 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech Co., Ltd) wasdissolved into 15 ml chloroform/ethanol (11:1, v/v), and added intowhich 500 ml human serum albumin solution (4% w/v) (CAS: 70024-90-7,Guangdong Shuanglin Biopharmaceutical. Co., Ltd.). The mixture wasemulsified for 2 min using a high shear disperser (Fluko FZ-20) toobtain a primary emulsion. The primary emulsion was then homogenizedusing a high pressure homogenizer (Model M110-EH30K, MFIC Company, USA)under a pressure of 10000-20000 psi to obtain a nano-emulsion.Subsequently, the nano-emulsion was transferred to a rotatory evaporator(Model R-210, Buchi Company, Switzerland) to remove the organic solventin the solution by vacuum evaporation at 40 mbar and at 40° C. in awater-bath. The paclitaxel-albumin nanoparticles were thus generatedwith an average diameter of 134 nm, and the suspension was translucent.

The suspension can be smoothly filtered through a 0.22 μm sterile filter(Sartorius AG, Germany). There was no significant variation of theparticle size after filtration, and no significant change was observedafter storage for 48 h at room temperature. The suspension was aliquotedand lyophilized for 24 h in a lyophilizer (Model LD-85S, Millrock, USA)to obtain a stable off-white cake.

Example 8

7.5 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech Co., Ltd) wasdissolved into 15 ml chloroform/ethanol (11:1, v/v), and added intowhich 500 ml human serum albumin solution (4% w/v) (CAS: 70024-90-7,Guangdong Shuanglin Biopharmaceutical. Co., Ltd.). The mixture wasemulsified for 2 min using a high shear disperser (Fluko FZ-20) toobtain a primary emulsion. The primary emulsion was then homogenizedusing a high pressure homogenizer (Model M110-EH30K, MFIC Company, USA)under a pressure of 10000-20000 psi to obtain a nano-emulsion.Subsequently, the nano-emulsion was transferred to a rotatory evaporator(Model R-210, Buchi Company, Switzerland) to remove the organic solventin the solution by vacuum evaporation at 40 mbar and at 40° C. in awater-bath. The paclitaxel-albumin nanoparticles were thus generatedwith an average diameter of 140 nm, and the suspension was translucent.

The suspension can be smoothly filtered through a 0.22 μm sterile filter(Sartorius AG, Germany). There was no significant variation of theparticle size after filtration, and no significant change was observedafter storage for 48 h at room temperature. The suspension was aliquotedand lyophilized for 24 h in a lyophilizer (Model LD-85S, Millrock, USA)to obtain a stable off-white cake.

Example 9

1 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech Co., Ltd) wasdissolved into 20 ml chloroform/ethanol (4:1, v/v), and added into which500 ml human serum albumin solution (2% w/v) (CAS: 70024-90-7, GuangdongShuanglin Biopharmaceutical. Co., Ltd.). The mixture was emulsified for2 min using a high shear disperser (Fluko FZ-20) to obtain a primaryemulsion. The primary emulsion was then homogenized using a highpressure homogenizer (Model M110-EH30K, MFIC Company, USA) under apressure of 10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator (Model R-210,Buchi Company, Switzerland) to remove the organic solvent in thesolution by vacuum evaporation at 40 mbar and at 40° C. in a water-bath.The paclitaxel-albumin nanoparticles were thus generated with an averagediameter of 136 nm, and the suspension was translucent.

The suspension can be smoothly filtered through a 0.22 μm sterile filter(Sartorius AG, Germany). There was no significant variation of theparticle size after filtration, and no significant change was observedafter storage for 48 h at room temperature. The suspension was aliquotedand lyophilized for 24 h in a lyophilizer (Model LD-85S, Millrock, USA)to obtain a stable off-white cake.

Example 10. Determination Methods for the Content of Human Serum Albuminand Paclitaxel

The content of human serum albumin was determined by HPLC. The humanserum albumin was determined at a wavelength of 228 nm in an HPLCequipped with a Tosohaas TSK G3000 SWXL gel column and a UV-detector(1260VWD G1314B, Agilent technologies), with a mobile phase of 0.1 mol/Ldipotassium hydrogen phosphate solution and an injection volume of 10μl. The albumin content was calculated using the external standardmethod.

Preparation of the test solutions: the test solutions were prepared bydiluting the solution for determination using 0.9% sodium chloridesolution to an albumin concentration lower than 3 mg/ml.

The content of paclitaxel was determined by HPLC. The paclitaxel wasdetermined at a wavelength of 228 nm in an HPLC equipped with a C18reverse phase column and a UV-detector (1260VWD G1314B, Agilenttechnologies), with a mobile phase of acetonitrile-water (1:1, v/v) andan injection volume of 10 μl. The paclitaxel content was calculatedusing the external standard method.

Preparation of the test solutions: the test solutions were prepared bydiluting the solution for determination using acetonitrile until fulldissolution of paclitaxel, with the concentration of 20-200 μg/ml.

Example 11

The product obtained in Example 1 was reconstituted in a 0.9% sodiumchloride solution to obtain sample 1, with the paclitaxel content of 5mg/ml. Subsequently, sample 1 was diluted using simulated blood plasmacontaining 5% albumin, so that the paclitaxel content may reach 20 μg/mlto obtain sample 2 (particles were completely disintegrated under suchcondition, and no paclitaxel-human serum albumin bound particles exist).1 ml sample 1 and 1 ml sample 2 were centrifuged at 21000×g for variousdurations, respectively. The concentrations of paclitaxel and albumin inthe supernatant were determined using the methods mentioned in Example10, and the results are listed in table 1.

TABLE 1 The concentrations of paclitaxel and albumin in the supernatantunder different centrifugation durations Centrifu- Sample 1 Sample 2gation 5 mg/ml solution 20 μg/ml solution duration Albumin PaclitaxelAlbumin Paclitaxel (min) (mg/ml) % (mg/ml) % (mg/ml) % (mg/ml) % 0 42.8100.0* 5.01 100.0* 52.7 100.0* 0.0217 100.0* 20 44.2 103.2 0.78 15.653.1 100.8 0.0218 99.5 40 45.2 105.6 0.38 7.7 52.2 99.0 0.0220 101.3 8044.9 104.9 0.11 2.1 53.5 101.6 0.0218 100.6 80 45.3** 105.8 0.11 2.153.5 101.5 0.0219 100.9 Note*: the percentage contents of albumin andpaclitaxel in supernatant were both calculated based on theconcentration at 0 min (100%). Note**: After reconstitution, theconcentration of albumin increased since the solution volume was reducedto 90% of its initial volume due to precipitation after centrifugationof the suspension.

It was suggested that no precipitation was generated aftercentrifugation for the sample fully disintegrated (Sample 2). Nosignificant variations of the paclitaxel and albumin concentrations insupernatant were observed for different centrifugation durations, i.e.,there was no paclitaxel crystal or heavy particle in the solution.

Precipitate occurred at the bottom after centrifugation for thenon-disintegrated sample (Sample 1). The paclitaxel concentration insupernatant decreased with centrifugation time, and finally reachedequilibrium. The albumin concentration slightly increased with time, andfinally reached equilibrium (the volume of the supernatant was about 90%of the total volume). In conclusion, the nanoparticles in the sample canbe isolated and purified by centrifugation.

After centrifugation for 60 min, the paclitaxel concentration insupernatant reached equilibrium. Thus, paclitaxel-albumin particles canbe isolated at 21000×g for 60 min.

Example 12. Preparation of the Nanoparticles

Using the centrifugation method mentioned in Example 11, particles wereisolated from the samples obtained in Example 1-9. After centrifugation,the supernatants were discarded, and the precipitates thus obtained werethe paclitaxel-albumin nanoparticles, which were referred as particle 1,2, 3, 4, 5, 6, 7, 8, and 9, corresponding to Example 1-9.

Example 13. Scanning Electron Microscopic Observation on Morphology ofthe Particles Before and after Separation

Lyophilized powder of the sample obtained in Example 5, and theprecipitate from Example 5 obtained in Example 12 (particle 5) wereobserved under a scanning electron microscope (S-4800, Hitachi). It canbe seen from the result in FIG. 1 that a small amount of particles inthe sample from Example 5 were embedded in the support agent formed by agreat amount of albumin. While for Particle 5, these particles existedindependently (See FIG. 2). It can thus be confirmed that theprecipitate obtained using the separation method in Example 11 was pureor substantially pure nanoparticles.

Example 14. The Ratio Between Albumin and Paclitaxel in the Particle

The paclitaxel-albumin nanoparticles (corresponding to Example 1-9)obtained in Example 12 were re-suspended respectively in 1 ml 0.9%sodium chloride solution. The contents of paclitaxel and albumin in thesamples were each determined using the method in Example 10, and theresults are as follows:

TABLE 2 The ratio between albumin and paclitaxel in various purifiednanoparticles obtained after centrifugation Concentration in oil AlbuminPaclitaxel phase content content (mg/ml) (mg/ml) (mg/ml)Albumin:Paclitaxel Example 8 500 0.24 5.58 0.043:1  Example 7 333 0.385.34 0.071:1  Example 6 167 0.65 5.03 0.13:1 Example 1 150 0.76 5.040.15:1 Example 5 125 0.83 5.16 0.16:1 Example 9 50 0.86 5.03 0.17:1Example 4 83 0.82 4.56 0.18:1 Example 3 42 1.11 4.63 0.24:1 Example 2 212.10 3.69 0.57:1

It has been suggested by the results above that the ratios betweenalbumin and paclitaxel in the purified nanoparticles from the productsobtained using different formulation process were different, and theyhave certain regularity. It also can be seen that increasedconcentration of paclitaxel in oil phase was inversely proportional tothe albumin content.

Example 15. Preparation of Purified Nanoparticles Using Dialysis

Dialysis was conducted in equal volume to the samples prepared inExample 2, Example 5, and Example 8 after reconstituted by water forinjection against the dialysis solution of 5% mannitol using aregenerated cellulose ultrafiltration membrane (PXC300C50, Millipore)with the cut-off molecular weight of 300K, and the dialysis fold was 5.The contents of paclitaxel and albumin in the samples were determinedafter dialysis using the method in Example 10, and the results are asfollows:

TABLE 3 The ratios between albumin and paclitaxel in the purifiednanoparticles from various formulations obtained by dialysis Albumincontent Paclitaxel content (mg/ml) (mg/ml) Albumin:Paclitaxel Example 80.25 5.12 0.048:1  Example 5 0.73 4.85 0.15:1 Example 2 2.19 3.71 0.59:1

The above results have indicated a substantially similar weight ratiobetween albumin and paclitaxel in the particles obtained by dialysisusing an ultrafiltration membrane, as compared to the particles obtainedby centrifugation. As a result, dialysis using an ultrafiltrationmembrane also can be used for isolating the particles in the suspensionfrom excessive albumin, and for replacement of the solution surroundingthe particles.

Example 16. Preparation of Purified Nanoparticles on ChromatographicColumn

20 ml Sepharose 4B gel was packed in a glass column with diameter of 10mm (Φ10 mm*230 mm, Beijing Mancang Technology Ltd.). The column wasequilibrated with 0.9% sodium chloride solution to 3 times of the columnvolumes. 1 ml samples prepared in Example 2, Example 5, and Example 8were loaded on the top of the gel column, and eluted using 0.9% sodiumchloride solution, respectively. The effluent was on-line monitoredusing a UV-detector at the wavelength of 280 nm. The effluentcorresponding to the first peak on the chromatogram was a slightlycloudy suspension, which was collected for determination of paclitaxeland albumin. Recording was continued until the second peak was finished.Both peaks were well separated, indicating that the particles and freealbumin can be effectively separated using the Sepharose 4B gel column.The contents of paclitaxel and albumin in the particles were determinedusing the method in Example 10, and the results are as follows:

TABLE 4 The ratios between albumin and paclitaxel in the purifiednanoparticles from various formulations obtained by separation on achromatographic column Albumin content Paclitaxel content (mg/ml)(mg/ml) Albumin:Paclitaxel Example 8 0.10 2.51 0.041:1  Example 5 0.332.33 0.14:1 Example 2 0.68 1.23 0.55:1

The above results indicated a substantially similar weight ratio betweenalbumin and paclitaxel in the particles obtained by separation on a gelcolumn, as compared to the particles obtained by centrifugation anddialysis. As a result, gel column separation also can be used forisolating the particles in the suspension from excessive albumin.

Example 17. Determination of the Free Albumin Content in the PurifiedNanoparticles Prepared by Dialysis Using Centrifugation

The particle suspension obtained by dialysis in Example 15 was furtherisolated by centrifugation under the conditions of Example 11. After allparticles were precipitated at the bottom of the centrifuge tube, thealbumin concentration in the supernatant was determined, and the resultsare listed in Table 5.

TABLE 5 The variation of albumin concentration in the purifiednanoparticles from various formulations obtained by dialysis before andafter centrifugation Concentration in the Concentration beforesupernatant after centrifugation (mg/ml) centrifugation (mg/ml) Example2 2.19 0.02 Example 5 0.73 0.01 Example 8 0.25 ND ND stands for lowerthan the limit of determination, i.e., not detected.

It can be seen from the above results that in the paclitaxel-albuminnanoparticle suspension obtained by dialysis, the proportion of freealbumin was quite low, and most albumin was bound to paclitaxel to formnanoparticles.

Example 18. Determination of the Free Albumin Content in the PurifiedNanoparticles Prepared by Chromatographic Column Separation UsingCentrifugation

The particle suspension obtained by chromatographic column separation inExample 16 was further subjected to centrifugation under the conditionsof Example 11. After all particles were precipitated at the bottom ofthe centrifuge tube, the albumin concentration in the supernatant wasdetermined, and the results are listed in Table 6.

TABLE 6 The variation of albumin concentration in the purifiednanoparticles from various formulations obtained by chromatographiccolumn separation before and after centrifugation Concentration in theConcentration before supernatant after centrifugation centrifugation(mg/ml) (mg/ml) Example 2 0.68 ND Example 5 0.33 ND Example 8 0.10 ND NDstands for lower than the limit of determination, i.e., not detected.

It can be seen from the above results that in the paclitaxel-albuminnanoparticle suspension obtained by chromatographic column separation,there was almost no free albumin, and most albumin was bound topaclitaxel to form nanoparticles.

Example 19. Determination of the Free Albumin Content in the PurifiedNanoparticles Prepared by Centrifugation Using Chromatographic ColumnSeparation

A particle suspension was obtained by re-suspending thepaclitaxel-albumin nanoparticles obtained in Example 12 in 0.9% sodiumchloride solution, and the purified nanoparticles and free albumin (ifany) were separated from each other using Sepharose 4B column asmentioned in Example 16. During elution, the UV absorbance curve wasmonitored. After the particles were completely eluted, the followingeluent was collected, in which the albumin concentration was determined.The results are listed in Table 7.

TABLE 7 The variation of albumin concentration in the purifiednanoparticles from various formulations obtained by centrifugationbefore and after chromatographic column separation Concentration beforeAlbumin concentration after column separation column separation (mg/ml)(mg/ml) Example 2 0.57 ND Example 5 0.16 ND Example 8 0.043 ND ND standsfor lower than the limit of determination, i.e., not detected.

It can be seen from the above results that in the paclitaxel-albuminnanoparticle suspension obtained by centrifugation, there was almost nofree albumin, and most albumin was bound to paclitaxel to formnanoparticles. In Example 17, 18, and 19, it can be demonstrated by theevidences observed in three isolation measures that purifiednanoparticles can be obtained by all three measures with almost no freealbumin.

Example 20. Determination of Particle Size and Potential

Paclitaxel-albumin nanoparticles obtained in Example 12 (correspondingto Example 1-9) were each re-suspended in purified water. The particlesize and the zeta potential of the purified nanoparticles were detectedusing Malven NANO-ZS laser particle sizer, and the results are listedbelow:

TABLE 8 The particle size and the potential of the purifiednanoparticles after centrifugation Average particle size Zeta potentialInitial average of the pure of the pure particle size particle particle(nm) (nm) (mV) Example 6 125 126.6 −28.7 Example 5 133 132 −27.1 Example7 134 127.5 −37 Example 1 136 137 −28.1 Example 9 136 137.4 −33.6Example 4 138 138.7 −34.2 Example 8 140 139 −39.4 Example 3 141 151.2−27.5 Example 2 145 145 −33

It can be seen from the results that the average particle size of thepurified nanoparticles obtained after centrifugation was substantiallythe same as its initial average particle size, and the zeta potentialwas still relatively high, so that the charge can be employed tostabilize the particle suspension.

Example 21. X Diffraction Pattern

The crystal form was determined in an X-ray diffraction instrument forthe drug substance of paclitaxel and lyophilized albumin powder, and theresults are shown in FIG. 3 and FIG. 4. As shown, the drug substance ofpaclitaxel was crystalline powder, and the lyophilized albumin powderwas amorphous powder. Representative purified nanoparticles obtained inExample 12 (corresponding to Example 2, 4, 6, and 8) were lyophilized ina lyophilizer to obtain solid powder, the crystal form of which wasdetected in an X-ray diffraction instrument, and the results are shownin FIGS. 5, 6, 7, and 8. It can be seen from the X diffraction patternthat the ratio of albumin to paclitaxel in the particles was in therange from 0.043:1 to 0.57:1, and both albumin and paclitaxel were inamorphous form, which was significantly different from the drugsubstance of paclitaxel, and slightly different from the lyophilizedalbumin powder.

Example 22. Release of Paclitaxel from the Particles

Using the separation method for particles established in Example 11,free component can be effectively separated from the component innanoparticle form. As a consequence, the release of paclitaxel fromparticles can be detected at different concentrations using such amethod, and specific procedure was shown below:

For the paclitaxel-albumin nanoparticles obtained in Example 12(corresponding to Example 1-9, respectively), representative purifiednanoparticles (corresponding to Example 2, 4, 6, and 8, and referred asParticle 2, 4, 6, and 8) were selected and 0.9% sodium chloride solutionwas added based on the ratio between albumin and paclitaxel, to form astock solution with paclitaxel concentration of 5 mg/ml. The stocksolution and simulated blood plasma solution were kept in 37° C. Thestock solution was diluted using simulated blood plasma solution toobtain a series of paclitaxel solutions with concentrations of 5000,1000, 200, 150, 100, 80, 50, 30, and 10 μg/ml.

Determination of release rate: the paclitaxel concentrations of theprepared sample solutions were determined, and at the same time, thepaclitaxel concentrations of the supernatants were also determined aftercentrifugation at 21000×g for 60 min using 1 ml of each sample. Therelease ratio of paclitaxel at each concentration was calculated bydividing the paclitaxel concentration in the supernatant by theconcentration before centrifugation, and the release curves were plottedand shown in FIG. 9 and FIG. 10. It can be seen from the release curvethat the ratio of albumin to paclitaxel in the particles was in therange from 0.043:1 to 0.057:1, and the release behaviors of paclitaxelfor the particles were substantially the same, all of which werecorrelated to the concentration.

Example 23. Preparation of Compositions Containing TherapeuticNanoparticles

Method 1 (Centrifugation-Re-suspension): therapeutic nanoparticlesobtained in Example 12 corresponding to Example 1-9 (referred asParticle 1, 2, 3, 4, 5, 6, 7, 8, and 9) were re-suspended in 5% mannitolsolution, 10% sucrose solution, 5% dextran solution, 10% lactosesolution, 10% trehalose solution, 10% maltose solution, 10% human serumalbumin solution, respectively, to establish paclitaxel concentrationsof 5 mg/ml. The re-suspensions were filtered through a 0.22 μm sterilefilter, and no significant variation of particle size was observed forthe particles in the filtrate. The suspensions were lyophilized in alyophilizer respectively to obtain the compositions containing thepharmaceutical nanoparticles.

Method 2 (Dialysis): the samples obtained in Example 1-9 were eachreconstituted to prepare suspensions with the paclitaxel concentrationof 5 mg/ml. Subsequently, the suspensions were dialyzed using a 300 kDaultrafiltration membrane respectively by 5% mannitol solution, 10%sucrose solution, 5% dextran solution, 10% lactose solution, 10% maltosesolution, 10% trehalose solution, and 10% human serum albumin solution.After dialyzing to 5-time of the initial volume, free human serumalbumin in the suspensions was all replaced. The suspensions werefiltered through a 0.22 μm sterile filter, and no significant variationof particle size was observed for the particles in the filtrate. Thesuspensions were lyophilized in a lyophilizer respectively to obtain thecompositions containing the therapeutic nanoparticles.

Example 24

The compositions containing the therapeutic nanoparticles obtained inMethod 1 (Centrifugation-Re-suspension) and Method 2 (Dialysis) inExample 23 were each reconstituted in water for injection to establish apaclitaxel concentration of 5 mg/ml. Stability for each solution wasobserved, and 12 h stability at room temperature was determined. Asshown, conventional lyoprotectants can play a protective role forpurified paclitaxel-albumin nanoparticles. The results are shown inTable 9 and Table 10:

TABLE 9 Stability of the compositions obtained byCentrifugation-Re-suspension Particles contained in the compositionParticle Particle Particle Particle Particle Particle Particle ParticleParticle 1 2 3 4 5 6 7 8 9 5% mannitol solution √ √ √ √ √ √ √ √ √ 10%sucrose solution √ √ √ √ √ √ √ √ √ 5% dextran solution √ √ √ √ √ √ √ √ √10% lactose solution √ √ √ √ √ √ √ √ √ 10% trehalose √ √ √ √ √ √ √ √ √solution 10% maltose solution √ √ √ √ √ √ √ √ √ 10% human serum √ √ √ √√ √ √ √ √ albumin solution Note: √ stands for a clear solution, with noobvious precipitate, and no variation of particle size as compared toits initial result.

TABLE 10 Stability of the compositions obtained by Dialysis Particlescontained in the composition Particle Particle Particle ParticleParticle Particle Particle Particle Particle 1 2 3 4 5 6 7 8 9 5%mannitol solution √ √ √ √ √ √ √ √ √ 10% sucrose solution √ √ √ √ √ √ √ √√ 5% dextran solution √ √ √ √ √ √ √ √ √ 10% lactose solution √ √ √ √ √ √√ √ √ 10% maltose solution √ √ √ √ √ √ √ √ √ 10% trehalose √ √ √ √ √ √ √√ √ solution 10% human serum √ √ √ √ √ √ √ √ √ albumin solution Note: √stands for a clear solution, with no obvious precipitate, and novariation of particle size as compared to its initial result.

As shown, the stability of particles can be maintained by replacing thealbumin surrounding the particles using Centrifugation-Re-suspension(Method 1) and Dialysis (Method 2).

Example 25. Sensitization Study in Guinea Pig

In this experiment, the product from Example 6 and 5% mannitolformulation comprising Particle 6 from Example 24 were selected as thetest drug, wherein the paclitaxel concentrations in the formulations arelisted in the table below. The sensitization dosages were selected as 3mg per guinea pig and 1.25 mg per guinea pig. Sensitization wasconducted by intraperitoneal injection once every other day, for a totalof 3 injections. At the same time, a positive control group (0.2%ovalbumin) and a negative control group (0.9% sodium chloride injection)were also established. Detailed dosage regimen is listed in the tablebelow.

TABLE 11 Protocol for sensitization study in guinea pig SensitizationExcitation Concentra- Adminis- Adminis- Adminis- Adminis- Number tion oftration tration tration tration of the the drug volume dosage volumedosage Group animal (mg/ml) (ml/guinea pig) (ml/guinea pig) (ml/guineapig) (ml/guinea pig) Negative 6 — 0.5 — 1 — control group Positive 6 20.5 1 1 2 control group Low dosage 6 2.5 0.5 1.25 1 2.5 of product fromExample 6 High dosage 6 6 0.5 3 1 6 of product from Example 6 Low dosage6 2.5 0.5 1.25 1 2.5 of Particle 6 High dosage 6 6 0.5 3 1 6 of Particle6

Administration method for sensitization: the back of guinea pig wasfirmly held by cup-shape left hand, allowing the abdominal skinstretched when the guinea pig was fixed. The abdomen of the guinea pigwas lifted, and the head was lowered down. After disinfecting of theinjection site by an alcohol wipe, the needle of a 2 ml disposablesyringe held by the right hand was punctured into the skin of the guineapig. The needle was inserted at the site 1 mm left to the midline oflower abdomen. When arriving at the subcutaneous part, the needle wasinserted forward for further 5 mm to 10 mm, and subsequently puncturedinto the abdominal cavity at an angle of 45°. After fixing the needle,the pharmaceutical solution was injected slowly. After the injection, adry cotton wipe was pressed on the pinprick in order to prevent theoutflow of the pharmaceutical. After 3 times of sensitization, theguinea pigs in the group of the formulation from Example 6 (3 mg/guineapig) became emaciated, and died. No abnormality was observed in othergroups.

Allergy excitation: excitation was conducted by intravenous injection,and the excitation was performed 10 d after the last sensitization withthe dosages of 6 mg/animal and 2.5 mg/animal.

Administration method for excitation: injection was performed to thelateral metatarsal vein of the guinea pig fixed by an assistant. Thestifles were grasped by the operator to fix the body of the animal. Itsvein was compressed, and the legs were in a stretched state. The hair atthe injection site was shaved (or the skin at the injection site wascut). After sterilization by alcohol wipes, thick lateral metatarsalvein can be seen. The needle of a 1 ml disposable syringe was puncturedinto the blood vessel along the direction to the heart by the righthand. After the injection, a dry cotton wipe was pressed on the pinprickin order to prevent bleeding.

The reaction of each animal and the time when allergy symptoms appearedor disappeared were observed immediately after the excitation for 30min. Maximal observation duration was 3 h. The results of allergicreaction are listed in Table 14.

TABLE 12 Symptoms of allergic reaction 0 Normal 1 Dysphoria 2Piloerection 3 Trembling 4 Nose scratching 5 Sneezing 6 Coughing 7Tachypnea 8 Urination 9 Defecation 10 Lacrimation 11 Dyspnea 12 Wheezing13 Peliosis 14 Gait disturbance 15 Jumping 16 Panting 17 Convulsion 18Rotation 19 Cheyne-stokes respiration 20 Death

TABLE 13 Evaluation criteria for systemic allergic reaction SymptomDegree Result  0 − Negative allergic reaction 1-4 + Weakly positiveallergic reaction  5-10 ++ Positive allergic reaction 11-19 +++ Stronglypositive allergic reaction 20 ++++ Extremely positive allergic reaction

TABLE 14 Results for active anaphylaxis of guinea pig Number Posi- ofthe Reaction level tive Group animal 1 2 3 4 5 6 rate Negative 6 − − − −− − − control group Positive 6 ++++ ++++ ++++ ++++ ++++ ++++ ++++control group Low dosage 6 + +++ +++ ++++ ++ ++ +++ of product fromExample 6 High dosage 6 / / / / / / NA* of product from Example 6 Lowdosage 6 − − − − − − − of Particle 6 High dosage 6 + − − + − + + ofParticle 6 *stands for the fact that the animal became emaciated anddead after sensitization.

It has been suggested by the results of active anaphylaxis in guineapigs that the nanoparticle formulation containing excessive albumin hasstronger sensitization, whereas the formulation containing purifiednanoparticles can significantly decrease the allergic reaction.

Example 26. Pharmacokinetic Study in Dogs

In this experiment, the product from Example 6 and 5% mannitolformulation comprising Particle 6 from Example 24 were selected as thetest drug, wherein the paclitaxel concentration in the formulations was5 mg/ml. Beagle dog was employed for the in vivo pharmacokinetic studiesof both formulations. In each group, 3 beagle dogs were tested withadministration dosage of 5 mg/kg, and administration time length of 30min. 2 ml blood was collected from the cephalic vein of the forelimb ofbeagle dogs immediately before administration (0 h), 15 min in theinfusion process (0.25 h since administration), the time point of needlewithdrawal (0.5 h since administration), and 0.58, 0.75, 1.0, 1.5, 2.5,3.5, 4.5, 6.5, 8.5, 24.5 h post administration, and each blood sampleswere placed into a heparin tube, shaken until homogenous, andcentrifuged at 3000 rpm for 10 min to obtain the plasma. The paclitaxelconcentration in plasma was determined by HPLC/MS/MS, and the drugconcentration was plotted versus time (See FIG. 11). It can be seen fromthe drug concentration-time plot that both formulations had almost thesame in vivo behaviors, so that there was no impact by excessive albuminon the in vivo behaviors of the particles.

Example 27. Allergic Symptoms During the Pharmacokinetic Study in Dogs

In the pharmacokinetic study in dogs conducted in Example 26, adversereactions were observed during the administration of both formulations.In the sample group of Example 6, various degrees of obvious allergicreactions appeared in 3 dogs, mainly including violently struggling,excessive salivation, erythema around mouth, and in individualexperimental animals, vomiting and urinary incontinence occurred duringadministration. While for the sample group of Particle 6, only slightlystruggling, salivation, and erythema around mouth appeared in part ofthe animals and the symptoms are mild. Consequently, the formulation ofpurified nanoparticles was capable of significantly alleviating theallergic reactions of the drug.

Example 28

The following samples were investigated for the maximum tolerated dose,the samples including the composition comprising the therapeuticnanoparticles using mannitol as the lyoprotectant (obtained from Method1 of Example 24, corresponding to the nanoparticles of Example 2, 6, and8, and referred as Particle 2, Particle 6, and Particle 8 below),product from Example 6, and Taxol® prepared with Cremophor.

Based on the recommendation of NCI of USA, modifications were made tothe experimental method for determining the maximum tolerated dose ofacute toxicity by single dose administration. For Particle 2, Particle 6and Particle 8, and lyophilized powder from Example 6, 400 mg/kg wasselected as the maximum administration dose, and the administration dosewas reduced in a ratio of 1.2, i.e., a series of doses of 400, 333, 278,231 and 193 mg/kg. For TAXOL®, 48 mg/kg was selected as the maximumadministration dose, and the administration dose was also reduced in aratio of 1.2, i.e., a series of doses of 48, 40, 33.3, 27.8, 23.1 and19.3 mg/kg. In each dose group, 3 KM male mice were assigned. Thegeneral conditions and variations of body weight of the animals wereobserved for 10 days after administration. If no animal died, noirreversible toxic response occurred, or no more than 15% of body weightloss appeared for 3 continuous days as compared to that of beforeexperiment during observation, the maximum administration dose wasconsidered as the maximum tolerated dose for the single administration.

The results indicated that the maximum tolerated dose of TAXOL® was 33.3mg/kg. In all administration groups, during administration of TAXOL®,the mice struggled violently, and coma and accelerated breathingoccurred after administration. In the dose groups of 40 mg/kg and 48mg/kg, cases of death occurred. In the rest dose groups, mice recoveredgradually after administration. The severity of toxicity symptoms andthe time demanded for recovery were correlated to the administrationdose. The maximum tolerated doses for Example 6 and Particle 2, Particle6 and Particle 8 were all 193 mg/kg. Adverse symptoms of the mice in thegroups of doses above 193 mg/kg mainly included body weight loss, dirtaround anus, rear limb weakness/paralysis, and death. The severity ofadverse symptoms was enhanced with increased administration dose. Therewas no obvious difference among the toxicities to the mice by Particle2, Particle 6, Particle 8 and the product from Example 6. However, allthe above toxicities were lower than that of TAXOL® prepared withCremophor.

Example 29. Inhibitory Effect on H22 Tumour of Liver Cancer in Mice

Particle 2, Particle 6, Particle 8, the product from Example 6, andTaxol® prepared with Cremophor were studied for liver cancer H22 tumorinhibitory effect in mice.

The animals were divided based on body weight. Ascitic cells of H22liver cancer were subcutaneously inoculated to the subcutaneous tissuein the axillary region of the forelimbs of male KM mice with theinoculation volume of 0.2 ml, which contained about 1.0×10⁶ cells. Theanimals were equally divided into 6 groups according to the inoculationtime.

Single intravenous administrations of Particle 2, Particle 6, Particle8, the product from Example 6 and Taxol® were performed at the maximumtolerated dose of mice 24 h after inoculation, i.e., the administrationdoses of Particle 2, Particle 6, Particle 8, and the product fromExample 6 were 193 mg/kg, and the administration dose of Taxol® was 33.3mg/kg. In the blank control group, 0.9% sodium chloride injection wasused for the single-intravenous administration. On the 12^(th) day afteradministration, the mice were sacrificed by CO₂ asphyxia, and the tumormass was taken out and weighed. The tumor inhibitory rate (%) can becalculated according to the following equation: Tumor inhibitoryrate=(1−average tumor weight in the test groups/average tumor weight inthe control group)×100%. The experimental results were statisticallyanalyzed by One-way ANOVA using statistical software of SPSS 19.0.

The experimental results are listed in Table 15. For single intravenousadministration at the maximum tolerated dose of mice, the growth of H22tumor was significantly inhibited in all groups. There was nointer-group difference in the tumor inhibitory effect of Particle 2,Particle 6, Particle 8 and the product from Example 6 on H22 tumor. Thetumor inhibitory rate of Particle 2, Particle 8 and the product fromExample 6 was significantly higher than TAXOL®.

TABLE 15 Effect on the body weight of mice bearing H22 tumor(n = 10, x ±sd) Initial Final Growth Tumor Tumor inhib- Dosage weight weight of bodyweight itory rate Group (mg/kg) (g) (g) weight (%) (g) (%) Particle 2193 22.8 ± 29.3 ± 28.9 0.7168 ± 70.9 1.6 2.7**^(▴▴) 0.5081**^(▴)Particle 6 193 23.9 ± 29.6 ± 24.0 0.9965 ± 59.6 1.5 2.1**^(▴▴) 0.6347**Particle 8 193 23.8 ± 29.0 ± 22.1 0.6090 ± 75.3 1.4 4.3**^(▴▴)0.4129**^(▴▴) The 193 23.7 ± 27.4 ± 15.8 0.7213 ± 70.7 product 1.42.3**^(▴▴) 0.4604**^(▴) from Example 6 Taxol ® 33.3 23.2 ± 35.1 ± 51.41.2789 ± 48.1 2.2 2.8* 0.5556* Control — 22.0 ± 38.3 ± 74.1 2.4644 ± 2.72.8 1.1753 **p < 0.01, *p < 0.05, as compared to the blank controlgroup: ^(▴▴)p < 0.01, ^(▴)p < 0.05, as compared to Taxol ®

Example 30. Inhibitory Effect on the Prostatic Cancer RM-1 Tumour ofMice

Particle 2, Particle 6, Particle 8, the product from Example 6, andTAXOL® prepared with Cremophor were studied for prostatic cancer RM-1tumor inhibitory effect in mice.

The RM-1 tumor cells at logarithmic growth phase were collected, and thecell number was adjusted to 2.5×10⁶ cells/ml. The suspension of tumorcells was inoculated to the subcutaneous tissue in the axillary regionof the forelimbs of 7 to 8-week-old C75 male mice with the inoculationvolume of 0.2 ml, which contained about 5×10⁵ cells. After inoculation,the remained suspension of tumor cells was counted under an opticalmicroscope, with the number of live tumor cells >95%. The mice weredivided into 6 groups based on inoculation time.

Single intravenous administrations of Particle 2, Particle 6, Particle8, the product from Example 6 and Taxol® were performed at the maximumtolerated dose of mice 72 h after inoculation, i.e., the administrationdoses of Particle 2, Particle 6, Particle 8, and the product fromExample 6 were 193 mg/kg, and the administration dose of Taxol® was 33.3mg/kg. In the blank control group, 0.9% sodium chloride injection wasused for the single-intravenous administration. After the outgrowth oftumor, the diameter was measured using a vernier caliper, and theanti-tumor effect of the pharmaceutical was observed dynamically. Thetumor volume (TV) can be calculated as follows: V=½×a×b², wherein a andb refer to the length and width of tumor, respectively. The tumor volumecan be calculated based on the results. The tumor volume inhibitory rate(%) can be calculated according to the following equation: Tumorinhibitory rate=(1−average tumor volume in the administrationgroups/average tumor volume in the control group)×100%. The experimentalresults were analyzed by statistical software SPSS 19.0. Theinter-measure tumor volume variation with time was analyzed by RepeatedMeasure Analysis, and the inter-group tumor volume variation among eachmeasure was analyzed by Multivariate.

The experimental results are listed in Table 16. Single intravenousadministrations were performed at the maximum tolerated dose 3 daysafter inoculation of prostatic cancer RM-1 tumor cells. As compared tothe blank control group, significant inhibitory effect on the growth ofthe prostatic cancer RM-1 tumor of mice was observed for Particle 2,Particle 6, Particle 8 and the product from Example 6, and no inhibitoryeffect on the tumor was seen for Taxol® prepared with Cremophor. Nosignificant difference in the inhibitory effect of Particle 2, Particle6, Particle 8 and the product from Example 6 was observed on RM-1 tumorof mice. However, as compared to Taxol®, all the above formulations hadsignificant inhibitory effect on the growth of tumor.

TABLE 16 Inhibitory effect on prostatic cancer RM-1 tumor of mice(n =10, x ± sd) Initial Final Growth Tumor volume (mm³)/Tumor inhibitoryrate % Dosage weight weight of body 7 d after 10 d after 12 d after 14 dafter (mg/kg) (g) (g) weight inoculation inoculation inoculationinoculation Particle 2 193 27.6 ± 32.3 ± 17.0% 107.9 ± 210.6 ± 471.6 ±715.5 ± 1.8 2.3 25.8**^(▴) 128.6*^(▴) 305.0 367.1^(▴) 59.7% 49.9% 28.9%40.7% Particle 6 193 29.4 ± 34.2 ± −6.3% 96.2 ± 122.6 ± 331.7 ± 600.3 ±2.0 3.9* 23.3**^(▴▴) 45.9**^(▴▴) 142.2*^(▴) 302.2*^(▴▴) 64.1% 70.8%50.0% 50.2% Particle 8 193 28.2 ± 326 ± 15.8% 107.1 ± 148.7 ± 385.1 ±736.4 ± 2.1 3.2 28.7**^(▴) 56.6**^(▴▴) 138.4* 472.9 60.0% 64.6% 41.9%38.9% The 193 28.1 ± 33.7 ± −9.9% 110.6 ± 187.1 ± 395.9 ± 590.0 ±product 2.0 2.6**^(▴) 18.2**^(▴) 60.0**^(▴) 147.8* 171.2*^(▴▴) from58.7% 55.5% 40.3% 51.1% Example 6 Taxol ® 33.3 26.8 ± 31.1 ± 16.2% 198.6± 430.3 ± 563.7 ± 1113.2 ± 1.9 2.7 103.9 272.7 311.3 460.0 25.8% −2.5%15.0%  7.7% Control 26.6 ± 30.5 ± 14.6% 267.6 ± 420.0 ± 663.2 ± 1205.7 ±1.5 2.3 169.1 217.7 338.4 735.1 **p < 0.01, *p < 0.05, as compared tothe blank control group: ^(▴▴)p < 0.01, ^(▴)p < 0.05, as compared toTaxol ®

Example 31. Inhibitory Effect on the Tumor of the Lewis Lung Cancer Mice

Particle 2, Particle 6, Particle 8, the product from Example 6, andTAXOL® prepared with Cremophor were studied for the inhibitory effect ontumor in Lewis lung cancer mice.

The Lewis tumor cells at logarithmic growth phase were collected, andthe cell number was adjusted to 2.5×10⁶ cells/ml. The suspension oftumor cells was inoculated to the subcutaneous tissue in the axillaryregion of the forelimbs of 7 to 8-week-old C57 male mice with theinoculation volume of 0.2 ml, which contained about 5×10⁵ cells.

The grouping, administration, indicator observation and statisticalmethod were the same as those in Example 18.

The experimental results are listed in Table 17. Single intravenousadministrations were performed at the maximum tolerated dose 3 daysafter inoculation of tumor cells of Lewis lung cancer mice. As comparedto the blank control group, significant inhibitory effect on the growthof the tumor of the Lewis lung cancer mice was observed for Particle 2,Particle 6, Particle 8 and the product from Example 6, and no inhibitoryeffect on the tumor was seen for Taxol® prepared with Cremophor. Nosignificant difference in the inhibitory effect of Particle 2, Particle6, Particle 8 and the product from Example 6 was observed on the tumorof the Lewis lung cancer mice. However, as compared to Taxol®, all theabove formulations had significant inhibitory effect on the growth oftumor.

TABLE 17 Inhibitory effect on the tumor of the Lewis lung cancer mice(n= 10, x ± sd) Initial weight g Growth Tumor volume (mm³)/Tumorinhibitory rate % Dosage Final weight of body 7 d after 10 d after 12 dafter 14 d after 17 d after 19 d after 21 d after Group mg/kg g weightinoculation inoculation inoculation inoculation inoculation inoculationinoculation Particle 2 193 25.7 ± 16.4% 45.5 ± 172.4 ± 166.0 ± 239.2 ±566.6 ± 856.9 ± 1608.1 ± 1.7 33.4**^(▴▴) 37.4**^(▴) 39.9**^(▴▴)88.8**^(▴▴) 273.9*^(▴) 436.5* 714.4 29.9 ± 67.7% 32.9% 48.1% 52.6% 48.5%45.9% 34.7% 2.3 Particle 6 193 26.4 ± 13.7% 11.7 ± 153.8 ± 177.7 ± 282.8± 640.1 ± 940.0 ± 1536.4 ± 1.2 25.1**^(▴▴) 70.1**^(▴) 119.0*^(▴▴)278.3^(▴) 701.1 788.6 1288.9 30.1 ± 91.7% 40.1% 44.5% 44.0% 41.9% 40.6%37.6% 1.8^(▴) Particle 8 193 26.7 ± 16.6% 0.0 ± 161.1 ± 157.4 ± 256.7 ±461.6 ± 850.7 ± 1278.3 ± 1.5 0.0**^(▴▴) 41.0**^(▴) 61.7**^(▴▴)146.3*^(▴▴) 244.3**^(▴) 1475.8* 507.3*^(▴▴) 31.1 ± 100.0%  37.3% 50.8%49.2% 58.1% 46.3% 48.1% 2.0^(▴▴) The 193 26.1 ± 21.2% 98.9 ± 196.5 ±210.7 ± 360.9 ± 671.7 ± 1030.0 ± 1539.8 ± product 1.5 41.2*^(▴) 46.872.8^(▴▴) 271.4 521.0 863.5 1342.5 from 31.6 ± 29.7% 23.5% 34.1% 28.6%  39% 34.9% 37.5% Example 6 2.0^(▴▴) Taxol ® 33.3 25.6 ± 9.6% 137.1 ±254.2 ± 373.8 ± 616.0 ± 1111.5 ± 1373.7 ± 2486.8 ± 1.8 35.3 108.6 139.5303.1 698.4 839.7 1148.3 28.0 ±  2.5%  1.0% −16.9%  −21.9%  −0.9% 13.2%−1.0% 2.2 Control 26.2 ± 14.3% 140.6 ± 256.8 ± 319.9 ± 505.2 ± 1101.2 ±1582.7 ± 2462.8 ± 2.2 39.1 83.1 149.3 251.2 555.6 833.5 1275.3 30.0 ±3.2 **p < 0.01, *p < 0.05, as compared to the blank control group:^(▴▴)p < 0.01, ^(▴)p < 0.05, as compared to Taxol ®

Example 32

As a commercially available pharmaceutical product, human serum albuminshould contain no more than 5% of the polymer according to its qualitystandard, since the albumin polymer may induce allergic reaction. Thedetermination method for albumin in Example 10 can distinguish albuminmonomer from the polymer. The albumin polymer and paclitaxel contentwere detected in the compositions from Example 1-9 (referred as initialformulation) and the compositions obtained from Method 1 of Example 24,respectively using mannitol and human serum albumin as a lyoprotectant(corresponding to Example 1-9), based on the determination method foralbumin and paclitaxel in Example 10, and the albumin polymer contentwas calculated per 1 mg paclitaxel. The results are listed in Table 18.

TABLE 18 Relative amount of albumin polymer and paclitaxel in variousformulations Initial Mannitol as Albumin as formulation lyoprotectantlyoprotectant (mg/mg) (mg/mg) (mg/mg) Example 1 1.7 0.03 0.18 Example 212.5 0.11 0.20 Example 3 6.3 0.05 0.19 Example 4 3.2 0.04 0.17 Example 52.1 0.03 0.18 Example 6 1.6 0.03 0.18 Example 7 0.8 ND 0.17 Example 80.9 ND 0.16 Example 9 2.0 0.03 0.17 Note: ND stands for lower than thelimit of determination, i.e., not detected

It has been suggested by the results that the content of albumin polymerin the product prepared directly using the method of prior art(Homogenization) was significantly higher than the content in thepost-added albumin of the composition by the preparation method of thepresent disclosure. This was resulted from albumin polymer newlygenerated in the homogenization process or evaporation process of theprior art. However, the content of albumin polymer in the formulationcomprising mannitol protectant was lower, since only very small amountof albumin was comprised in the formulation, so that the total amountwas lower than the amount of the polymer in initial formulation.

Example 33. Recovering Albumin for Preparation of the Nanoparticles

The dialysate resulted from the dialysis in Example 15 was collected,and concentrated to an albumin content of about 4% using a regeneratedcellulose ultrafiltration membrane with a cut-off molecular weight of10K. The concentrate was dialyzed in equal volume by purified water.After dialyzed to a 5-time volume, 4% albumin solution can be recovered.

According to the method in Example 5, paclitaxel-albumin nanoparticleswere prepared using recovered albumin solution. Consequently, theaverage diameter of the prepared paclitaxel-albumin nanoparticles was134 nm, and the suspension was translucent, which was similar to theproduct obtained in Example 5.

The suspension can be smoothly filtered through a 0.22 μm sterilefilter. There was no significant variation of the particle size afterfiltration, and no significant change was observed for the suspensionafter storage for 48 h at room temperature. The suspension was aliquotedand lyophilized for 24 h in a lyophilizer to obtain a stable off-whitecake.

Example 34. Preparation of Purified Docetaxel-Albumin Particles

3 g docetaxel was dissolved into 15 ml chloroform/ethanol (1:1, v/v),and added into 500 ml human serum albumin solution (4% w/v). The mixturewas emulsified for 2 min using a high shear disperser (Fluko FZ-20) toobtain a primary emulsion. The primary emulsion was then homogenizedusing a high pressure homogenizer under a pressure of 10000-20000 psi toobtain a nano-emulsion. Subsequently, the nano-emulsion was transferredto a rotatory evaporator to remove the organic solvent in the solutionby vacuum evaporation at 40 mbar and at 40° C. in a water-bath. Thedocetaxel-albumin nanoparticles were thus generated with an averagediameter of 110-150 nm, and the suspension was translucent.

The nanoparticles were isolated from the resultant docetaxel-albuminnanoparticle suspension by centrifugation method referred in Example 11at 21000×g for 60 min. The supernatant was discarded, and the purifiednanoparticles were collected. Purified nanoparticles were re-suspendedin 5% mannitol solution. The re-suspension was filtered through a 0.22μm sterile filter, and there was no significant variation of theparticle size in the filtrate. The content of docetaxel in the solutionwas determined using HPLC. Based on the content, the solution wassubsequently aliquoted into vials at an amount of 50 mg per vial. Thevials were placed in a lyophilizer and lyophilized for 48 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and docetaxelcontent in the product, the ratio between albumin and docetaxel can becalculated as 0.1:1.

Example 35. Preparation of Purified 2′-O-Hexanoyldocetaxel AlbuminParticles

695 mg 2′-O-hexanoyldocetaxel was dissolved into 6 ml chloroform/ethanol(10:1, v/v), and added into 102 ml human serum albumin solution (10%w/v). The mixture was emulsified for 2 min using a high shear disperser(Fluko FZ-20) to obtain a primary emulsion. The primary emulsion wasthen homogenized using a high pressure homogenizer under a pressure of10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator to remove theorganic solvent in the solution by vacuum evaporation at 40 mbar and at40° C. in a water-bath. The 2′-O-hexanoyldocetaxel albumin nanoparticleswere thus generated with an average diameter of 75-100 nm, and thesuspension was translucent.

The nanoparticles were isolated from the resultant2′-O-hexanoyldocetaxel albumin nanoparticle suspension by dialysismethod against 5% mannitol solution referred in Example 15. The obtainedpurified nanoparticles suspension was filtered through a 0.22 μm sterilefilter, and there was no significant variation of the particle size inthe filtrate. The content of 2′-O-hexanoyldocetaxel in the solution wasdetermined using HPLC. Based on the content, the solution wassubsequently aliquoted into vials at an amount of 50 mg per vial. Thevials were placed in a lyophilizer and lyophilized for 48 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and2′-O-hexanoyldocetaxel content in the product, the ratio between albuminand 2′-O-hexanoyldocetaxel can be calculated as 0.27:1.

Example 36. Preparation of Purified 2′-Benzoyl Docetaxel AlbuminParticles

226 mg 2′-benzoyl docetaxel was dissolved into 2 ml chloroform/ethanol(9:1, v/v), and added into 35 ml human serum albumin solution (5% w/v).The mixture was emulsified for 2 min using a high shear disperser (FlukoFZ-20) to obtain a primary emulsion. The primary emulsion was thenhomogenized using a high pressure homogenizer under a pressure of18000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator to remove theorganic solvent in the solution by vacuum evaporation at 40 mbar and at40° C. in a water-bath. The 2′-benzoyl docetaxel albumin nanoparticleswere thus generated with an average diameter of 30-60 nm, and thesuspension was translucent.

The nanoparticles were isolated from the resultant 2′-benzoyl docetaxelalbumin nanoparticle suspension by chromatographic column separationmethod referred in Example 16. 10% lactose solution was used asdialysate. The obtained purified nanoparticles suspension was filteredthrough a 0.22 μm sterile filter, and there was no significant variationof the particle size in the filtrate. The content of 2′-benzoyldocetaxel in the solution was determined using HPLC. Based on thecontent, the solution was subsequently aliquoted into vials at an amountof 50 mg per vial. The vials were placed in a lyophilizer andlyophilized for 65 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and 2′-benzoyldocetaxel content in the product, the ratio between albumin and2′-benzoyl docetaxel can be calculated as 0.95:1.

Example 37. Preparation of Purified Rapamycin Albumin Particles

166 mg rapamycin was dissolved into 1 ml chloroform/ethanol (11:1, v/v),and added into 37 ml human serum albumin solution (5% w/v). The mixturewas emulsified for 2 min using a high shear disperser (Fluko FZ-20) toobtain a primary emulsion. The primary emulsion was then homogenizedusing a high pressure homogenizer under a pressure of 10000-20000 psi toobtain a nano-emulsion. Subsequently, the nano-emulsion was transferredto a rotatory evaporator to remove the organic solvent in the solutionby vacuum evaporation at 40 mbar and at 40° C. in a water-bath. Therapamycin nanoparticles were thus generated with an average diameter of50-85 nm, and the suspension was translucent.

The nanoparticles were isolated from the resultant rapamycin albuminnanoparticle suspension by chromatographic column separation methodreferred in Example 16. 5% dextrane solution was used as dialysate. Theobtained purified nanoparticles suspension was filtered through a 0.22μm sterile filter, and there was no significant variation of theparticle size in the filtrate. The content of rapamycin in the solutionwas determined using HPLC. Based on the content, the solution wassubsequently aliquoted into vials at an amount of 10 mg per vial. Thevials were placed in a lyophilizer and lyophilized for 48 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and rapamycincontent in the product, the ratio between albumin and rapamycin can becalculated as 0.63:1.

Example 38. Preparation of Purified Temsirolimus Albumin Particles

101 mg temsirolimus was dissolved into 0.5 ml chloroform/ethanol (7:1,v/v), and added into 19.5 ml human serum albumin solution (5% w/v). Themixture was emulsified for 2 min using a high shear disperser (FlukoFZ-20) to obtain a primary emulsion. The primary emulsion was thenhomogenized using a high pressure homogenizer under a pressure of10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator to remove theorganic solvent in the solution by vacuum evaporation at 40 mbar and at40° C. in a water-bath. The temsirolimus nanoparticles were thusgenerated with an average diameter of 80-115 nm, and the suspension wastranslucent.

The nanoparticles were isolated from the resultant temsirolimus albuminnanoparticles by centrifugation method referred in Example 11 at 21000×gfor 60 min. The supernatant was discarded, and the purifiednanoparticles were collected. Purified nanoparticles were re-suspendedin 5% mannitol solution. The re-suspension was filtered through a 0.22μm sterile filter, and there was no significant variation of theparticle size in the filtrate. The content of temsirolimus in thesolution was determined using HPLC. Based on the content, the solutionwas subsequently aliquoted into vials at an amount of 10 mg per vial.The vials were placed in a lyophilizer and lyophilized for 48 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin andtemsirolimus content in the product, the ratio between albumin andtemsirolimus can be calculated as 0.16:1.

Example 39. Preparation of Purified Everolimus Albumin Particles

78 mg everolimus was dissolved into 1 ml chloroform/ethanol (9:1, v/v),and added into 22 ml human serum albumin solution (8% w/v). The mixturewas emulsified for 2 min using a high shear disperser (Fluko FZ-20) toobtain a primary emulsion. The primary emulsion was then homogenizedusing a high pressure homogenizer under a pressure of 10000-20000 psi toobtain a nano-emulsion. Subsequently, the nano-emulsion was transferredto a rotatory evaporator to remove the organic solvent in the solutionby vacuum evaporation at 40 mbar and at 40° C. in a water-bath. Theeverolimus nanoparticles were thus generated with an average diameter of80-110 nm, and the suspension was translucent.

The nanoparticles were isolated from the resultant everolimus albuminnanoparticle suspension by dialysis method referred in Example 15against 5% dextrane. The obtained purified nanoparticles suspension wasfiltered through a 0.22 μm sterile filter, and there was no significantvariation of the particle size in the filtrate. The content ofeverolimus in the solution was determined using HPLC. Based on thecontent, the solution was subsequently aliquoted into vials at an amountof 10 mg per vial. The vials were placed in a lyophilizer andlyophilized for 60 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and everolimuscontent in the product, the ratio between albumin and everolimus can becalculated as 0.32:1.

Example 40. Preparation of Purified Romidepsin Albumin Particles

113 mg romidepsin was dissolved into 2 ml chloroform/ethanol (8:1, v/v),and added into 35 ml human serum albumin solution (10% w/v). The mixturewas emulsified for 2 min using a high shear disperser (Fluko FZ-20) toobtain a primary emulsion. The primary emulsion was then homogenizedusing a high pressure homogenizer under a pressure of 10000-20000 psi toobtain a nano-emulsion. Subsequently, the nano-emulsion was transferredto a rotatory evaporator to remove the organic solvent in the solutionby vacuum evaporation at 40 mbar and at 40° C. in a water-bath. The 2romidepsin nanoparticles were thus generated with an average diameter of120-150 nm, and the suspension was translucent.

The nanoparticles were isolated from the resultant romidepsin albuminnanoparticle suspension by chromatographic column separation methodreferred in Example 16. 10% sucrose solution was used as dialysate. Theobtained purified nanoparticles suspension was filtered through a 0.22μm sterile filter, and there was no significant variation of theparticle size in the filtrate. The content of romidepsin in the solutionwas determined using HPLC. Based on the content, the solution wassubsequently aliquoted into vials at an amount of 50 mg per vial. Thevials were placed in a lyophilizer and lyophilized for 48 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and romidepsincontent in the product, the ratio between albumin and romidepsin can becalculated as 0.25:1.

Example 41. Preparation of Purified Pirarubicin Albumin Particles

147 mg pirarubicin was dissolved into 0.75 ml chloroform/ethanol (5:1,v/v), and added into 19.5 ml human serum albumin solution (5% w/v). Themixture was emulsified for 2 min using a high shear disperser (FlukoFZ-20) to obtain a primary emulsion. The primary emulsion was thenhomogenized using a high pressure homogenizer under a pressure of10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator to remove theorganic solvent in the solution by vacuum evaporation at 40 mbar and at40° C. in a water-bath. The 2 pirarubicin nanoparticles were thusgenerated with an average diameter of 100-120 nm, and the suspension wastranslucent.

The nanoparticles were isolated from the resultant pirarubicin albuminnanoparticle suspension by dialysis method against 5% mannitol solutionreferred in Example 15. The obtained purified nanoparticles werefiltered through a 0.22 μm sterile filter, and there was no significantvariation of the particle size in the filtrate. The content ofpirarubicin in the solution was determined using HPLC. Based on thecontent, the solution was subsequently aliquoted into vials at an amountof 10 mg per vial. The vials were placed in a lyophilizer andlyophilized for 48 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and pirarubicincontent in the product, the ratio between albumin and pirarubicin can becalculated as 0.47:1.

Example 42. Preparation of Purified Aclacinomycin Albumin Particles

135 mg aclacinomycin was dissolved into 2 ml chloroform/ethanol (8:1,v/v), and added into 25 ml human serum albumin solution (8% w/v). Themixture was emulsified for 2 min using a high shear disperser (FlukoFZ-20) to obtain a primary emulsion. The primary emulsion was thenhomogenized using a high pressure homogenizer under a pressure of10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator to remove theorganic solvent in the solution by vacuum evaporation at 40 mbar and at40° C. in a water-bath. The aclacinomycin nanoparticles were thusgenerated with an average diameter of 80-150 nm, and the suspension wastranslucent.

The nanoparticles were isolated from the resultant aclacinomycin albuminnanoparticle suspension by chromatographic column separation methodreferred in Example 16. 5% mannitol solution was used as dialysate. There-suspension was filtered through a 0.22 μm sterile filter, and therewas no significant variation of the particle size in the filtrate. Thecontent of aclacinomycin in the solution was determined using HPLC.Based on the content, the solution was subsequently aliquoted into vialsat an amount of 10 mg per vial. The vials were placed in a lyophilizerand lyophilized for 48 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin andaclacinomycin content in the product, the ratio between albumin andaclacinomycin can be calculated as 0.13:1.

Example 43. Preparation of Purified Cabazitaxel Albumin Particles

289 mg cabazitaxel was dissolved into 3 ml chloroform/ethanol (10:1,v/v), and added into 67 ml human serum albumin solution (5% w/v). Themixture was emulsified for 2 min using a high shear disperser (FlukoFZ-20) to obtain a primary emulsion. The primary emulsion was thenhomogenized using a high pressure homogenizer under a pressure of10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator to remove theorganic solvent in the solution by vacuum evaporation at 40 mbar and at40° C. in a water-bath. The cabazitaxel nanoparticles were thusgenerated with an average diameter of 65-85 nm, and the suspension wastranslucent.

The nanoparticles were isolated from the resultant cabazitaxel albuminnanoparticle suspension by equal volume dialysis referred in Example 15against 5% mannitol. The purified nanoparticles suspension obtained werefiltered through a 0.22 μm sterile filter, and there was no significantvariation of the particle size in the filtrate. The content ofcabazitaxel in the solution was determined using HPLC. Based on thecontent, the solution was subsequently aliquoted into vials at an amountof 50 mg per vial. The vials were placed in a lyophilizer andlyophilized for 55 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and cabazitaxelcontent in the product, the ratio between albumin and cabazitaxel can becalculated as 0.27:1.

Example 44. Preparation of Purified Amiodardone Albumin Particles

90 mg amiodardone was dissolved into 2 ml chloroform/ethanol (11:1,v/v), and added into 58 ml human serum albumin solution (8% w/v). Themixture was emulsified for 2 min using a high shear disperser (FlukoFZ-20) to obtain a primary emulsion. The primary emulsion was thenhomogenized using a high pressure homogenizer under a pressure of10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator to remove theorganic solvent in the solution by vacuum evaporation at 40 mbar and at40° C. in a water-bath. The 2 amiodardone nanoparticles were thusgenerated with an average diameter of 70-105 nm, and the suspension wastranslucent.

The nanoparticles were isolated from the resultant amiodardone albuminnanoparticle suspension by centrifugation method referred in Example 11at 21000×g for 60 min. The supernatant was discarded, and the purifiednanoparticles were collected. Purified nanoparticles were re-suspendedin 5% dextran solution. The re-suspension was filtered through a 0.22 μmsterile filter, and there was no significant variation of the particlesize in the filtrate. The content of amiodardone in the solution wasdetermined using HPLC. Based on the content, the solution wassubsequently aliquoted into vials at an amount of 10 mg per vial. Thevials were placed in a lyophilizer and lyophilized for 57 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and amiodardonecontent in the product, the ratio between albumin and amiodardone can becalculated as 0.43:1.

Example 45. Preparation of Purified Liothyronine Albumin Particles

93 mg liothyronine was dissolved into 2 ml chloroform/ethanol (9:1,v/v), and added into 46 ml human serum albumin solution (8% w/v). Themixture was emulsified for 2 min using a high shear disperser (FlukoFZ-20) to obtain a primary emulsion. The primary emulsion was thenhomogenized using a high pressure homogenizer under a pressure of10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator to remove theorganic solvent in the solution by vacuum evaporation at 40 mbar and at40° C. in a water-bath. The 2 liothyronine nanoparticles were thusgenerated with an average diameter of 85-125 nm, and the suspension wastranslucent.

The nanoparticles were isolated from the resultant liothyronine albuminnanoparticle suspension by centrifugation method referred in Example 11at 21000×g for 60 min. The supernatant was discarded, and the purifiednanoparticles were collected. Purified nanoparticles were re-suspendedin 5% mannitol solution. The re-suspension was filtered through a 0.22μm sterile filter, and there was no significant variation of theparticle size in the filtrate. The content of liothyronine in thesolution was determined using HPLC. Based on the content, the solutionwas subsequently aliquoted into vials at an amount of 10 mg per vial.The vials were placed in a lyophilizer and lyophilized for 50 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin andliothyronine content in the product, the ratio between albumin andliothyronine can be calculated as 0.39:1.

Example 46. Preparation of Purified Epothilone B Albumin Particles

127 mg Epothilone B was dissolved into 2 ml chloroform/ethanol (10:1,v/v), and added into 52 ml human serum albumin solution (5% w/v). Themixture was emulsified for 2 min using a high shear disperser (FlukoFZ-20) to obtain a primary emulsion. The primary emulsion was thenhomogenized using a high pressure homogenizer under a pressure of10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator to remove theorganic solvent in the solution by vacuum evaporation at 40 mbar and at40° C. in a water-bath. The Epothilone B nanoparticles were thusgenerated with an average diameter of 80-115 nm, and the suspension wastranslucent.

The nanoparticles were isolated from the resultant Epothilone B albuminnanoparticle suspension by equal volume dialysis against 5% mannitol.The purified nanoparticles suspension obtained was filtered through a0.22 μm sterile filter, and there was no significant variation of theparticle size in the filtrate. The content of Epothilone B in thesolution was determined using HPLC. Based on the content, the solutionwas subsequently aliquoted into vials at an amount of 10 mg per vial.The vials were placed in a lyophilizer and lyophilized for 65 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and EpothiloneB content in the product, the ratio between albumin and Epothilone B canbe calculated as 0.14:1.

Example 47. Preparation of Purified 10-Hydroxy Camptothecin AlbuminParticles

93 mg 10-hydroxy camptothecin was dissolved into 2 ml chloroform/ethanol(10:1, v/v), and added into 48 ml human serum albumin solution (5% w/v).The mixture was emulsified for 2 min using a high shear disperser (FlukoFZ-20) to obtain a primary emulsion. The primary emulsion was thenhomogenized using a high pressure homogenizer under a pressure of10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator to remove theorganic solvent in the solution by vacuum evaporation at 40 mbar and at40° C. in a water-bath. The 10-hydroxy camptothecin nanoparticles werethus generated with an average diameter of 100-135 nm, and thesuspension was translucent.

The nanoparticles were isolated from the resultant 10-hydroxycamptothecin albumin nanoparticle suspension by centrifugation methodreferred in Example 11 at 21000×g for 60 min. The supernatant wasdiscarded, and the purified nanoparticles were collected. Purifiednanoparticles were re-suspended in 5% mannitol solution. There-suspension was filtered through a 0.22 μm sterile filter, and therewas no significant variation of the particle size in the filtrate. Thecontent of 10-hydroxy camptothecin in the solution was determined usingHPLC. Based on the content, the solution was subsequently aliquoted intovials at an amount of 20 mg per vial. The vials were placed in alyophilizer and lyophilized for 72 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and 10-hydroxycamptothecin content in the product, the ratio between albumin and10-hydroxy camptothecin can be calculated as 0.26:1.

Example 48. Preparation of Purified Cyclosporine Albumin Particles

148 mg cyclosporine was dissolved into 1.5 ml chloroform/ethanol (11:1,v/v), and added into 18.5 ml human serum albumin solution (4% w/v). Themixture was emulsified for 2 min using a high shear disperser (FlukoFZ-20) to obtain a primary emulsion. The primary emulsion was thenhomogenized using a high pressure homogenizer under a pressure of10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator to remove theorganic solvent in the solution by vacuum evaporation at 40 mbar and at40° C. in a water-bath. The cyclosporine nanoparticles were thusgenerated with an average diameter of 100-135 nm, and the suspension wastranslucent.

The nanoparticles were isolated from the resultant cyclosporine albuminnanoparticle suspension by chromatographic column separation methodreferred in Example 16. 10% lactose was use as the dialysate. Thepurified nanoparticles suspension obtained was filtered through a 0.22μm sterile filter, and there was no significant variation of theparticle size in the filtrate. The content of cyclosporine in thesolution was determined using HPLC. Based on the content, the solutionwas subsequently aliquoted into vials at an amount of 50 mg per vial.The vials were placed in a lyophilizer and lyophilized for 72 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin andcyclosporine content in the product, the ratio between albumin andcyclosporine can be calculated as 0.26:1.

Example 49. Preparation of Purified Tanespimycin Albumin Particles

88 mg tanespimycin was dissolved into 2 ml chloroform/ethanol (8:1,v/v), and added into 18.5 ml human serum albumin solution (10% w/v). Themixture was emulsified for 2 min using a high shear disperser (FlukoFZ-20) to obtain a primary emulsion. The primary emulsion was thenhomogenized using a high pressure homogenizer under a pressure of10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator to remove theorganic solvent in the solution by vacuum evaporation at 40 mbar and at40° C. in a water-bath. The tanespimycin nanoparticles were thusgenerated with an average diameter of 85-105 nm, and the suspension wastranslucent.

The nanoparticles were isolated from the resultant tanespimycin albuminnanoparticle suspension by chromatographic column separation methodreferred in Example 16. 5% mannitol was use as the dialysate. Thepurified nanoparticles suspension was filtered through a 0.22 μm sterilefilter, and there was no significant variation of the particle size inthe filtrate. The content of tanespimycin in the solution was determinedusing HPLC. Based on the content, the solution was subsequentlyaliquoted into vials at an amount of 50 mg per vial. The vials wereplaced in a lyophilizer and lyophilized for 72 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin andtanespimycin content in the product, the ratio between albumin andtanespimycin can be calculated as 0.62:1.

Example 50. Preparation of Purified Propofol Albumin Particles

603 mg propofol was dissolved into 6 ml chloroform, and added into 73 mlhuman serum albumin solution (10% w/v). The mixture was emulsified for 2min using a high shear disperser (Fluko FZ-20) to obtain a primaryemulsion. The primary emulsion was then homogenized using a highpressure homogenizer under a pressure of 10000-20000 psi to obtain anano-emulsion. Subsequently, the nano-emulsion was transferred to arotatory evaporator to remove the organic solvent in the solution byvacuum evaporation at 40 mbar and at 40° C. in a water-bath. Thepropofol nanoparticles were thus generated with an average diameter of70-115 nm, and the suspension was translucent.

The nanoparticles were isolated from the resultant propofol albuminnanoparticle suspension by centrifugation method referred in Example 11at 21000×g for 60 min. The supernatant was discarded, and the purifiednanoparticles were collected. Purified nanoparticles were re-suspendedin 10% lactose solution. The re-suspension was filtered through a 0.22μm sterile filter, and there was no significant variation of theparticle size in the filtrate. The content of propofol in the solutionwas determined using HPLC. Based on the content, the solution wassubsequently aliquoted into vials at an amount of 50 mg per vial. Thevials were placed in a lyophilizer and lyophilized for 72 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and propofolcontent in the product, the ratio between albumin and propofol can becalculated as 0.58:1.

Example 51. Preparation of Purified Vinblastine Sulfate AlbuminParticles

1.25 g vinblastine sulfate was dissolved into 10 mlchloroform/isopropanol (10:1, v/v), and added into 202 ml human serumalbumin solution (5% w/v). The mixture was emulsified for 2 min using ahigh shear disperser (Fluko FZ-20) to obtain a primary emulsion. Theprimary emulsion was then homogenized using a high pressure homogenizerunder a pressure of 10000-20000 psi to obtain a nano-emulsion.Subsequently, the nano-emulsion was transferred to a rotatory evaporatorto remove the organic solvent in the solution by vacuum evaporation at40 mbar and at 40° C. in a water-bath. The vinblastine sulfatenanoparticles were thus generated with an average diameter of 90-130 nm,and the suspension was translucent.

The nanoparticles were isolated from the resultant vinblastine sulfatealbumin nanoparticle suspension by equal volume dialysis against 5%mannitol. The purified nanoparticles suspension obtained was filteredthrough a 0.22 μm sterile filter, and there was no significant variationof the particle size in the filtrate. The content of vinblastine sulfatein the solution was determined using HPLC. Based on the content, thesolution was subsequently aliquoted into vials at an amount of 50 mg pervial. The vials were placed in a lyophilizer and lyophilized for 72 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and vinblastinesulfate content in the product, the ratio between albumin andvinblastine sulfate can be calculated as 0.23:1.

Example 52. Preparation of Purified Exemestane Albumin Particles

155 mg exemestane was dissolved into 3 ml chloroform/ethanol (6:1, v/v),and added into 27 ml human serum albumin solution (5% w/v). The mixturewas emulsified for 2 min using a high shear disperser (Fluko FZ-20) toobtain a primary emulsion. The primary emulsion was then homogenizedusing a high pressure homogenizer under a pressure of 10000-20000 psi toobtain a nano-emulsion. Subsequently, the nano-emulsion was transferredto a rotatory evaporator to remove the organic solvent in the solutionby vacuum evaporation at 40 mbar and at 40° C. in a water-bath. Theexemestane nanoparticles were thus generated with an average diameter of70-100 nm, and the suspension was translucent.

The nanoparticles were isolated from the resultant exemestane albuminnanoparticle suspension by equal volume dialysis against 5% mannitol.The purified nanoparticles suspension obtained was filtered through a0.22 μm sterile filter, and there was no significant variation of theparticle size in the filtrate. The content of exemestane in the solutionwas determined using HPLC. Based on the content, the solution wassubsequently aliquoted into vials at an amount of 50 mg per vial. Thevials were placed in a lyophilizer and lyophilized for 72 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and exemestanecontent in the product, the ratio between albumin and exemestane can becalculated as 0.19:1.

Example 53. Preparation of Purified Flutamide Albumin Particles

189 mg flutamide was dissolved into 2 ml chloroform/tert-butanol (6:1,v/v), and added into 38 ml human serum albumin solution (8% w/v). Themixture was emulsified for 2 min using a high shear disperser (FlukoFZ-20) to obtain a primary emulsion. The primary emulsion was thenhomogenized using a high pressure homogenizer under a pressure of10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator to remove theorganic solvent in the solution by vacuum evaporation at 40 mbar and at40° C. in a water-bath. The flutamide nanoparticles were thus generatedwith an average diameter of 100-120 nm, and the suspension wastranslucent.

The nanoparticles were isolated from the resultant flutamide albuminnanoparticle suspension by chromatographic column separation methodreferred in Example 16. 5% dextran solution was used as dialysate. Thepurified nanoparticles suspension obtained was filtered through a 0.22μm sterile filter, and there was no significant variation of theparticle size in the filtrate. The content of flutamide in the solutionwas determined using HPLC. Based on the content, the solution wassubsequently aliquoted into vials at an amount of 50 mg per vial. Thevials were placed in a lyophilizer and lyophilized for 60 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and flutamidecontent in the product, the ratio between albumin and flutamide can becalculated as 0.35:1.

Example 54. Preparation of Purified Fulvestrant Albumin Particles

350 mg fulvestrant was dissolved into 3 ml dichloromethane/ethanol (6:1,v/v), and added into 67 ml human serum albumin solution (5% w/v). Themixture was emulsified for 2 min using a high shear disperser (FlukoFZ-20) to obtain a primary emulsion. The primary emulsion was thenhomogenized using a high pressure homogenizer under a pressure of10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator to remove theorganic solvent in the solution by vacuum evaporation at 40 mbar and at40° C. in a water-bath. The fulvestrant nanoparticles were thusgenerated with an average diameter of 105-150 nm, and the suspension wastranslucent.

The nanoparticles were isolated from the resultant fulvestrant albuminnanoparticle suspension by chromatographic column separation methodreferred in Example 16. 10% lactose was used as dialysate. The purifiednanoparticles suspension obtained was filtered through a 0.22 μm sterilefilter, and there was no significant variation of the particle size inthe filtrate. The content of fulvestrant in the solution was determinedusing HPLC. Based on the content, the solution was subsequentlyaliquoted into vials at an amount of 20 mg per vial. The vials wereplaced in a lyophilizer and lyophilized for 60 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and fulvestrantcontent in the product, the ratio between albumin and fulvestrant can becalculated as 0.29:1.

Example 55. Preparation of Purified Semustine Albumin Particles

650 mg semustine was dissolved into 5 ml chloroform/ethanol (6:1, v/v),and added into 89 ml human serum albumin solution (4% w/v). The mixturewas emulsified for 2 min using a high shear disperser (Fluko FZ-20) toobtain a primary emulsion. The primary emulsion was then homogenizedusing a high pressure homogenizer under a pressure of 10000-20000 psi toobtain a nano-emulsion. Subsequently, the nano-emulsion was transferredto a rotatory evaporator to remove the organic solvent in the solutionby vacuum evaporation at 40 mbar and at 40° C. in a water-bath. Thesemustine nanoparticles were thus generated with an average diameter of85-120 nm, and the suspension was translucent.

The nanoparticles were isolated from the resultant semustine albuminnanoparticle suspension by centrifugation method referred in Example 11at 21000×g for 60 min. The supernatant was discarded, and the purifiednanoparticles were collected. Purified nanoparticles were re-suspendedin 5% mannitol solution. The re-suspension was filtered through a 0.22μm sterile filter, and there was no significant variation of theparticle size in the filtrate. The content of semustine in the solutionwas determined using HPLC. Based on the content, the solution wassubsequently aliquoted into vials at an amount of 20 mg per vial. Thevials were placed in a lyophilizer and lyophilized for 70 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and semustinecontent in the product, the ratio between albumin and semustine can becalculated as 0.58:1.

Example 56. Preparation of Purified Thiocolchicine Dimer AlbuminParticles

456 mg thiocolchicine dimer was dissolved into 5 ml chloroform/ethanol(9:1, v/v), and added into 58 ml human serum albumin solution (8% w/v).The mixture was emulsified for 2 min using a high shear disperser (FlukoFZ-20) to obtain a primary emulsion. The primary emulsion was thenhomogenized using a high pressure homogenizer under a pressure of10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator to remove theorganic solvent in the solution by vacuum evaporation at 40 mbar and at40° C. in a water-bath. The thiocolchicine dimer nanoparticles were thusgenerated with an average diameter of 65-90 nm, and the suspension wastranslucent.

The nanoparticles were isolated from the resultant thiocolchicine dimeralbumin nanoparticle suspension by chromatographic column separationmethod referred in Example 16. 10% lactose was used as dialysate. Thepurified nanoparticles suspension obtained was filtered through a 0.22μm sterile filter, and there was no significant variation of theparticle size in the filtrate. The content of thiocolchicine dimer inthe solution was determined using HPLC. Based on the content, thesolution was subsequently aliquoted into vials at an amount of 20 mg pervial. The vials were placed in a lyophilizer and lyophilized for 70 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin andthiocolchicine dimer content in the product, the ratio between albuminand thiocolchicine dimer can be calculated as 0.37:1.

Example 57. Preparation of Purified Ibuprofen Dimer Albumin Particles

348 mg ibuprofen was dissolved into 3 ml dichloromethane/ethanol (11:1,v/v), and added into 62 ml human serum albumin solution (5% w/v). Themixture was emulsified for 2 min using a high shear disperser (FlukoFZ-20) to obtain a primary emulsion. The primary emulsion was thenhomogenized using a high pressure homogenizer under a pressure of10000-20000 psi to obtain a nano-emulsion. Subsequently, thenano-emulsion was transferred to a rotatory evaporator to remove theorganic solvent in the solution by vacuum evaporation at 40 mbar and at40° C. in a water-bath. The ibuprofen nanoparticles were thus generatedwith an average diameter of 65-95 nm, and the suspension wastranslucent.

The nanoparticles were isolated from the resultant ibuprofen albuminnanoparticle suspension by chromatographic column separation methodreferred in Example 16. 5% dextran was used as dialysate. The purifiednanoparticles suspension obtained was filtered through a 0.22 μm sterilefilter, and there was no significant variation of the particle size inthe filtrate. The content of ibuprofen in the solution was determinedusing HPLC. Based on the content, the solution was subsequentlyaliquoted into vials at an amount of 20 mg per vial. The vials wereplaced in a lyophilizer and lyophilized for 50 h.

When the lyophilized product was reconstituted in water for injection,the resultant cake was dissolved rapidly, the suspension wastranslucent, and no significant variation of the particle size wasobserved. After determination of the human serum albumin and ibuprofencontent in the product, the ratio between albumin and ibuprofen can becalculated as 0.19:1.

Example 58. Albumin Concentration Influences Inhibition Effect of Drugson Human Tumor Cells

Human lung cancer cell SPC-A-1 and breast cancer cell MCF-7 were seededto 96-well plate, cultured overnight and made them adherent to thewells. Then, the medium was changed to serum-free medium in order tostarve the cells. Example 6, particle 6 and Abraxane were added to thecells at final concentrations of 160, 320, and 640 μg/ml (paclitaxelconcentration) for 24 hours at 37° C. Cell inhibition rate of differentdosing groups was compared by using MTT method, and the results wereshown in the following table and FIGS. 12 and 13. Moreover, inhibitionrate-drug concentration curve were drew too. As showing in theinhibition rate-drug concentration curve, the inhibition rate ofparticle 6 was significantly higher than other two groups, and nosignificant difference between Example 6 and Abaxane. That meansredundant albumin will reduce the inhibition effect of paclitaxel ontumor cells.

TABLE 19 Cells inhibition rate of different treatment InhibitionParticle 6 Exemple 6 Abraxane rate % 640 μg/ml 320 μg/ml 160 μg/ml 640μg/ml 320 μg/ml 160 μg/ml 640 μg/ml 320 μg/ml 160 μg/ml MCF-7 cells No.1 60.12 74.97 24.00 43.23 22.50 −3.81 25.90 10.70 6.20 No. 2 69.59 71.1236.91 30.74 11.14 1.71 42.30 15.78 −8.14 No. 3 68.46 58.84 37.87 46.656.63 20.53 39.88 11.30 −1.85 No. 4 69.78 70.09 38.65 52.02 23.66 5.7345.50 2.42 −0.08 No. 5 71.69 72.93 43.91 46.87 22.18 13.55 47.26 5.39−4.86 No. 6 71.97 57.52 50.47 25.65 30.17 −7.71 31.19 22.88 −1.64 mean68.60 67.58 38.63 40.86 19.38 5.00 38.67 11.41 −1.73 SD 3.98 6.83 8.029.43 7.99 9.70 7.68 6.69 4.41 SPC-A-1 cells No. 1 66.49 25.07 16.2930.99 5.24 9.25 31.75 11.40 −3.20 No. 2 62.32 31.29 −3.14 20.50 −11.75−3.19 24.09 7.84 −12.89 No. 3 64.18 25.75 0.91 29.13 3.97 −11.90 13.089.58 4.11 No. 4 70.26 32.73 −12.03 32.69 7.03 −5.39 18.25 2.98 −4.37 No.5 67.98 33.58 9.87 25.88 19.83 12.75 30.02 6.42 −1.04 No. 6 77.24 44.837.26 28.46 10.29 29.57 18.94 14.17 12.62 mean 68.08 32.21 3.19 27.945.77 5.18 22.69 8.73 −0.80 SD 4.82 6.52 9.22 3.94 9.40 13.80 6.63 3.577.84

Example 59. Albumin Concentration Influences Uptake of Drugs by HumanVascular Endothelial Cells

Human umbilical vascular endothelial cells EA.hy 926 were seeded at8×10⁵ to 6-well plate. Before adding drugs, the medium was changed intoserum-free medium. Drugs was divided into 5 groups, group A was particle6, B was Example 6, C was Abraxane, D was particle 6+0.5% HSA monomer, Ewas particle 6+0.5% HSA polymer. Different groups of drug were added tothe cells at final concentrations of 50, 100, and 200 μg/ml (paclitaxelconcentration) for 2 hours at 37° C. After 2 hours, the cells werewashed by PBS for 3 times, then 500 ul 5% TritonX-100 was added to eachwells to lyse the cells. The paclitaxel concentration in cell lysis wasdetected by the method mentioned in example 10, and the results wereshown in the following table and FIG. 14. The results show that cellularuptake of paclitaxel of group A was higher than group B and group C, andof group B and C had no significant difference. Furthermore, group D washigher than group E. That means higher HSA concentration hindered druguptake to vascular endothelial cells, and compared to monomer, the HSApolymer would be more impede the cellular uptake.

TABLE 20 Drug concentration in cells of different treatment μg/ml 50 100200 Particle 6 concentration 2.367 4.102 5.864 value 2.165 3.873 6.2272.668 4.217 6.531 mean 2.400 4.064 6.207 SD 0.253 0.175 0.334 Exemple 6concentration 1.362 2.415 3.374 value 1.626 2.517 3.668 1.654 2.6883.941 mean 1.547 2.540 3.661 SD 0.161 0.138 0.284 Abraxane concentration1.462 2.246 3.861 value 1.623 2.522 3.521 1.711 2.613 4.031 mean 1.5992.460 3.804 SD 0.126 0.191 0.260 Particle 6 + concentration 1.221 2.1683.055 0.5% HSA value 1.254 2.517 3.251 monomer 1.021 2.231 3.461 mean1.165 2.305 3.256 SD 0.126 0.186 0.203 Particle 6 + concentration 0.7641.533 1.988 0.5% HSA value 0.966 1.687 1.845 polymer 0.878 1.921 2.356mean 0.869 1.714 2.063 SD 0.101 0.195 0.264

Example 60

The contents of albumin and paclitaxel (PTX) and the ratio of polymer inthe human serum albumin used, product obtained in Example 6, and thecorresponding nanoparticle 6 were determined using the method in Example10. The results are listed in the following table.

TABLE 21 Contents of albumin polymer (Poly-HSA) Monomer PolymerAlbumin/palictaxel PolyHSA/PTX (%) (%) (mg/mg) (mg/mg) HSA 95.8 4.2 — —Example 6 80.7 19.3 8.8 1.7 Particle 6 73.5 26.5 0.13 0.034

It has been revealed by the results that the ratio of poly-HSA wassignificantly increased during the preparation process of paclitaxelalbumin nanoparticles. However, in the product prepared by thepreparation method of the present disclosure, the amount of poly-HSA inunit weight is significantly reduced because of the decrease of totalalbumin content.

The amount of polymer in an albumin solution was increased by dialysisusing a regenerated cellulose ultrafiltration membrane (PXC100C50,Millipore) with the cut-off molecular weight of 100K. The ratio ofalbumin polymer was detected using the method in Example 10, and thecontent of albumin was determined by Kjeldahl method to avoid thedeviation produced by the different responses to potential polymers ofHPLC method.

TABLE 22 The content of albumin polymer in HSA before and after dialysisMonomer (%) Polymer (%) HSA before dialysis 95.8 4.2 HSA after dialysis25.1 74.9

It can be seen from the result that the albumin solution with a majorityamount of polymer can be obtained by dialysis process.

Example 61. Sensitization Study of Poly-HSA in Guinea Pigs

In this experiment, the HSA and poly-HSA obtained from Example 60 wereselected as the test drug. The dosages were selected as 1 mg per guineapig and 0.3 mg per guinea pig. Sensitization was conducted byintraperitoneal injection once every other day, for a total of 3injections. At the same time, a positive control group (0.2% ovalbumin)and a negative control group (0.9% sodium chloride injection) were alsoestablished. Detailed dosage regimen is listed in the table below.

TABLE 23 Protocol for sensitization study in guinea pig sensitizationExcitation Concentra- Adminis- Adminis- Adminis- Adminis- Number tion oftration tration tration tration of the drug volume dosage volume dosageGroup animals (mg/ml) (ml/animal) (mg/animal) (ml/animal) (mg/animal)Negative 6 — 0.5 — 1 — control group Positive 6 2 0.5 1 1 2 controlgroup Low dosage 6 0.6 0.5 0.3 1 0.6 of HSA High dosage 6 2 0.5 1 1 2 ofHSA Low dosage 6 0.6 0.5 1.3 1 0.6 of Poly- HSA High dosage 6 2 0.5 1 12 of Poly- HSA

Administration method for sensitization: the back of guinea pig wasfirmly held by cup-shape left hand, allowing the abdominal skinstretched when the guinea pig was fixed. The abdomen of the guinea pigwas lifted, and the head was lowered down. After disinfecting of theinjection site by an alcohol wipe, the needle of a 2 ml disposablesyringe held by the right hand was punctured into the skin of the guineapig. The needle was inserted at the site 1 mm left to the midline oflower abdomen. When arriving at the subcutaneous part, the needle wasinserted forward for further 5 mm to 10 mm, and subsequently puncturedinto the abdominal cavity at an angle of 45°. After fixing the needle,the pharmaceutical solution was injected slowly. After the injection, adry cotton wipe was pressed on the pinprick in order to prevent theoutflow of the pharmaceutical.

Allergy excitation: excitation was conducted by intravenous injection,and the excitation was performed 10 d after the last sensitization withthe dosages of 2 mg/animal and 0.6 mg/animal.

Administration method for excitation: injection was performed to thelateral metatarsal vein of the guinea pig fixed by an assistant. Thestifles were grasped by the operator to fix the body of the animal. Itsvein was compressed, and the legs were in a stretched state. The hair atthe injection site was shaved (or the skin at the injection site wascut). After sterilization by alcohol wipes, thick lateral metatarsalvein can be seen. The needle of a 1 ml disposable syringe was puncturedinto the blood vessel along the direction to the heart by the righthand. After the injection, a dry cotton wipe was pressed on the pinprickin order to prevent bleeding.

The reaction of each animal and the time when allergy symptoms appearedor disappeared were observed immediately after the excitation for 30min. Maximal observation duration was 3 h. The results of allergicreaction are listed in the following table.

TABLE 24 Symptoms of allergic reaction 0 Normal 1 Dysphoria 2Piloerection 3 Trembling 4 Nose scratching 5 Sneezing 6 Coughing 7Tachypnea 8 Urination 9 Defecation 10 Lacrimation 11 Dyspnea 12 Wheezing13 Peliosis 14 Gait disturbance 15 Jumping 16 Panting 17 Convulsion 18Rotation 19 Cheyne-stokes respiration 20 Death

TABLE 25 Evaluation criteria for systemic allergic reaction SymptomDegree Result  0 − Negative allergic reaction 1-4 + Weakly positiveallergic reaction  5-10 ++ Positive allergic reaction 11-19 +++ Stronglypositive allergic reaction 20 ++++ Extremely positive allergic reaction

TABLE 26 Results for active anaphylaxis of guinea pig Number Posi- ofthe Reaction level tive Group animal 1 2 3 4 5 6 rate Negative 6 − − − −− − − control group Positive 6 ++++ ++++ ++++ ++++ ++++ ++++ ++++control group Low dosage 6 + − − − + − + of HSA High dosage 6 + + ++++ + + ++ of HSA Low dosage 6 +++ ++++ +++ ++ +++ +++ +++ of Poly- HSAHigh dosage 6 ++++ ++++ ++++ ++++ ++++ ++++ ++++ of Poly- HSA

The results show that the samples containing more poly-HSA had strongersensitization at same level of total HSA content.

Example 62

2.5 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech Co., Ltd) wasdissolved into 15 ml chloroform/ethanol (11:1, v/v), and added intowhich 500 ml human serum albumin solution (4% w/v) (CAS: 70024-90-7,Guangdong Shuanglin Biopharmaceutical. Co., Ltd.). The mixture wasemulsified for 2 min using a high shear disperser (Fluko FZ-20) toobtain a primary emulsion. The primary emulsion was then homogenizedusing a high pressure homogenizer (Model M110-EH30K, MFIC Company, USA)under a pressure of 10000-20000 psi to obtain a nano-emulsion.Subsequently, the nano-emulsion was transferred to a rotatory evaporator(Model R-210, Buchi Company, Switzerland) to remove the organic solventin the solution by vacuum evaporation at 40 mbar and at 40° C. in awater-bath. The paclitaxel-albumin nanoparticles were thus generatedwith an average diameter of 129 nm, and the suspension was translucent.The suspension can be smoothly filtered through a 0.22 μm sterile filter(Sartorius AG, Germany). There was no significant variation of theparticle size after filtration, and no significant change was observedafter storage for 48 h at room temperature.

The purified nanoparticles were isolated respectively by centrifugationin Example 11, by dialysis in Example 15 and by chromatographic columnseparation in Example 16. The results showed that the suspension becameturbid and precipitated during dialysis. Thus purified nanoparticlescouldn't be obtained though this method. While the purifiednanoparticles obtained by centrifugation and chromatographic columnseparation were turbid and precipitated in 60 min.

Example 63

2.5 g paclitaxel (CAS: 33069-62-4, Yunnan Hande Bio-Tech Co., Ltd) wasdissolved into 15 ml chloroform/ethanol (11:1, v/v), and added intowhich 500 ml human serum albumin solution (4% w/v) (CAS: 70024-90-7,Guangdong Shuanglin Biopharmaceutical. Co., Ltd.). The mixture wasemulsified for 2 min using a high shear disperser (Fluko FZ-20) toobtain a primary emulsion. The primary emulsion was then homogenizedusing a high pressure homogenizer (Model M110-EH30K, MFIC Company, USA)under a pressure of 10000-20000 psi to obtain a nano-emulsion.Subsequently, the nano-emulsion was transferred to a rotatory evaporator(Model R-210, Buchi Company, Switzerland) to remove the organic solventin the solution by vacuum evaporation at 40 mbar and at 40° C. in awater-bath. The paclitaxel-albumin nanoparticles were thus generatedwith an average diameter of 128 nm, and the suspension was translucent.The suspension can be smoothly filtered through a 0.22 μm sterile filter(Sartorius AG, Germany). There was no significant variation of theparticle size after filtration, and no significant change was observedafter storage for 48 h at room temperature.

Dialysis was conducted in equal volume to the samples prepared againstpurified water using a regenerated cellulose ultrafiltration membrane(PXC300C50, Millipore) with the cut-off molecular weight of 30K, and thedialysis fold was 5.

The purified nanoparticles were isolated respectively by centrifugationin Example 11, by dialysis in Example 15 and by chromatographic columnseparation in Example 16. The resulted translucent suspensions from allthe 3 methods were stable and can be smoothly filtered through a 0.22 μmsterile filter (Sartorius AG, Germany). There was no significantvariation of the particle size after filtration, and no significantchange was observed after storage for 48 h at room temperature

Comparing Examples 62 and 63, an extra process of equal volume dialysisagainst purified water was conducted in Example 63 to remove theresidual solvents. The removal of the residual solvents improvedstability of suspension of purified nanoparticles.

Discussion:

Prior art formulations have a high ratio of albumin to an activeingredient (for example, 9:1). It has been unexpectedly discovered bythe present inventors that in such a formulation, most albumin acts onlyas a protective or support agent in the lyophilized product. Most drugmolecules (for example, paclitaxel) are encapsulated in thenanoparticles, and albumin that does not form particles is substantiallyfree of drug molecules. In prior art formulations, most human serumalbumin molecules are not bound to the drug molecules.

After administration, particles in prior art formulations aredisintegrated rapidly, and complexes are formed between the drugmolecules and the endogenous human serum albumin. Consequently,excessive human serum albumin in the prior art formulations does notcontribute to the efficacy of the formulations (see Example 26), butinstead causes safety risks due to its aggregation and immunogenicity(see Examples 32, 25 and 27). Furthermore, extra albumin may competewith the drug-albumin complex to bind to the gp60 receptor, thusdecrease the drug uptake by the vascular endothelial cells (see Example59). Moreover, after binding to the poly-HSA formed in preparationprocess, the gp60 receptor may be unable to be transcytosed effectively,thus preventing the receptor from being released. This may furtherinhibit the drug uptake by the endothelial cells, and thus decrease theeffective concentration of drugs.

Based on the discoveries above, purified nanoparticles of the presentdisclosure have a lower albumin: active ingredient ratio. It has beenconfirmed by the present inventors that only a small amount of humanserum albumin was required for forming stable nanoparticles with drugmolecules. The reduction in human serum albumin in purifiednanoparticles and compositions thereof provided herein reduces adversereactions associated with aggregation and immunogenicity of human serumalbumin.

After administration, purified nanoparticles provided herein were ableto be disintegrated rapidly, and bound to endogenous albumin forcirculation in vivo. There were no significantly difference observed interms of safety and efficacy in animal studies compared with prior artformulation. However, purified nanoparticles and compositions thereofprovided herein improved uptake by human vascular endothelial cells aswell as delivery and effectiveness of active ingredients in thenanoparticles to or in human target cells.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

The invention claimed is:
 1. Purified therapeutic nanoparticles,consisting essentially of paclitaxel and human serum albumin (HSA),wherein the weight ratio of HSA to paclitaxel in the therapeuticnanoparticles is from 0.03:1 to 0.95:1, wherein less than 10% HSA in thetherapeutic nanoparticles is free HSA that is not incorporated into thenanoparticles, wherein the therapeutic nanoparticles contain less than0.05 mg/ml organic solvent(s), and wherein when suspended in 0.9% sodiumchloride or 5% mannitol, the resulting suspension is stable for 48 hoursat room temperature.
 2. A pharmaceutical composition, comprising thepurified therapeutic nanoparticles of claim 1, wherein less than 10% ofHSA in the composition is free HSA that is not incorporated in thenanoparticles.
 3. The pharmaceutical composition of claim 2, wherein theweight ratio of HSA to paclitaxel of the therapeutic nanoparticles isfrom 0.03:1 to 0.7:1, and wherein the average particle size of thetherapeutic nanoparticles is from 50 nm to 190 nm.
 4. The pharmaceuticalcomposition according to claim 2, wherein the pharmaceutical compositionis in the form of liquid or lyophilized powder.
 5. The pharmaceuticalcomposition according to claim 2, wherein the pharmaceutical compositionalso comprises lyophilization excipient when the pharmaceuticalcomposition is in the form of lyophilized powder.
 6. The pharmaceuticalcomposition according to claim 5, wherein the lyophilization excipientis selected from one or more of mannitol, sucrose, lactose, maltose,trehalose, and dextran.
 7. The pharmaceutical composition according toclaim 2, wherein at most 5% of the HSA in the composition is free HSA(by weight) that is not incorporated in the nanoparticles.
 8. Thepharmaceutical composition according to claim 2, wherein the weightratio of HSA to paclitaxel of the therapeutic particles is from 0.03:1to 0.7:1.
 9. The pharmaceutical composition according to claim 2,wherein the therapeutic nanoparticles contain less than 5 ug/ml organicsolvent(s).
 10. The pharmaceutical composition according to claim 2,wherein less than 1% of the HSA in the composition is free HSA (byweight) that is not incorporated in the nanoparticles.
 11. Thepharmaceutical composition according to claim 2, wherein the averageparticle size of the therapeutic nanoparticles is from 100 nm to 160 nm.12. The pharmaceutical composition of claim 2, wherein the weight ratioof HSA to paclitaxel is from 0.10:1 to 0.6:1.
 13. The pharmaceuticalcomposition according to claim 2, wherein the organic solvent(s) is apure solvent having low water-solubility and a low boiling point or amixture of a solvent having low water-solubility and a low boiling pointwith a small molecular alcohol.
 14. The pharmaceutical compositionaccording to claim 2, wherein the weight ratio of HSA to paclitaxel intherapeutic nanoparticles is from 0.04:1 to 0.75:1.
 15. Thepharmaceutical composition according to claim 2, wherein the averageparticle size of the therapeutic nanoparticles is from 30 to 200 nm. 16.A method for preparing the purified therapeutic nanoparticles of claim1, comprising: 1) dissolving paclitaxel in organic solvent to form anoil phase, and dissolving human serum albumin in water to form anaqueous phase; 2) forming an oil-in-water emulsion using the oil phaseand aqueous phase of step 1); 3) removing the organic solvent in theemulsion to obtain a suspension containing the therapeuticnanoparticles; and 4) removing free HSA that is not incorporated in thenanoparticles from the suspension to obtain purified therapeuticnanoparticles.
 17. The method according to claim 16, wherein the organicsolvent is selected from one or more of chloroform and ethanol.
 18. Themethod according to claim 16, further comprising: between steps 3) and4), a step of dialyzing the suspension of step 3) with an aqueoussolution to remove remaining organic solvent from the suspension. 19.The method according to claim 18, wherein the aqueous solution is water.20. The method according to claim 16, wherein said separating in step 4)is conducted using a method selected from: centrifugation, dialysis, andexclusion chromatography.
 21. A method for preparing the pharmaceuticalcomposition according to claim 2, comprising: 1) dissolving paclitaxelin organic solvent to form an oil phase, and dissolving human serumalbumin in water to form an aqueous phase; 2) forming an oil-in-wateremulsion using the oil phase and aqueous phase of step 1); 3) removingthe organic solvent in the emulsion to obtain a suspension containingthe therapeutic nanoparticles; 4) removing free HSA that is notincorporated in the nanoparticles to obtain purified therapeuticnanoparticles; 5) re-suspending the purified therapeutic nanoparticlesin an excipient-containing solution; and 6) optionally lyophilizing there-suspension of the purified therapeutic nanoparticles to obtain thepharmaceutical composition.
 22. The method of claim 21, furthercomprising: between steps 3) and 4), a step of dialyzing the suspensionof step 3) with an aqueous solution to remove remaining organic solventfrom the suspension.
 23. The method according to claim 22, wherein theaqueous solution is water.
 24. A method for preparing the pharmaceuticalcomposition according to claim 2, comprising: 1) dissolving paclitaxelin organic solvent to form an oil phase, and dissolving human serumalbumin in water to form an aqueous phase; 2) forming an oil-in-wateremulsion using the oil phase and aqueous phase of step 1); 3) removingthe organic solvent in the emulsion to obtain a suspension containingthe therapeutic nanoparticles; 4) dialyzing the suspension obtainedafter removal of the organic solvent by an excipient-containing solutionto remove free HSA that is not incorporated in the nanoparticles; and 5)optionally lyophilizing the dialyzed suspension to obtain thepharmaceutical composition.
 25. The method of claim 24, furthercomprising: between steps 3) and 4), a step of dialyzing the suspensionof step 3) with an aqueous solution to remove remaining organic solventfrom the suspension.
 26. The method of claim 25, wherein the aqueoussolution is water.
 27. A method for treating cancer, comprising:administering a therapeutically effective amount of the pharmaceuticalcomposition according to claim 2 to a subject in need thereof.
 28. Amethod for treating cancer, comprising: administering a therapeuticallyeffective amount of the pharmaceutical composition according to claim 3to a subject in need thereof.